Brassica GAT event and compositions and methods for the identification and/or detection thereof

ABSTRACT

Compositions and methods related to transgenic glyphosate tolerant  Brassica  plants are provided. Specifically, the present invention provides  Brassica  plants having a DP-073496-4 event which imparts tolerance to glyphosate. The  Brassica  plant harboring the DP-073496-4 event at the recited chromosomal location comprises genomic/transgene junctions within SEQ ID NO: 2 or with genomic/transgene junctions as set forth in SEQ ID NO: 12 and/or 13. The characterization of the genomic insertion site of the event provides for an enhanced breeding efficiency and enables the use of molecular markers to track the transgene insert in the breeding populations and progeny thereof. Various methods and compositions for the identification, detection, and use of the event are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This Application is a Divisional of U.S. application Ser. No.12/953,852, filed Nov. 24, 2010, now allowed, which claims the benefitof U.S. Provisional Application No. 61/309,385, filed Mar. 1, 2010, andU.S. Provisional Application No. 61/263,923, filed Nov. 24, 2009, whichis herein incorporated by reference in their entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of 398263seqlist.txt, a creation date of Nov. 24, 2010, and a sizeof 40 Kb. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

FIELD OF THE INVENTION

This invention is in the field of molecular biology. More specifically,this invention pertains to expression of a sequence that conferstolerance to glyphosate.

BACKGROUND OF THE INVENTION

The expression of foreign genes in plants is known to be influenced bytheir location in the plant genome, perhaps due to chromatin structure(e.g., heterochromatin) or the proximity of transcriptional regulatoryelements (e.g., enhancers) close to the integration site (Weising, etal., (1988) Ann. Rev. Genet. 22:421-477). At the same time the presenceof the transgene at different locations in the genome influences theoverall phenotype of the plant in different ways. For this reason, it isoften necessary to screen a large number of events in order to identifyan event characterized by optimal expression of an introduced gene ofinterest. For example, it has been observed in plants and in otherorganisms that there may be a wide variation in levels of expression ofan introduced gene among events. There may also be differences inspatial or temporal patterns of expression, for example, differences inthe relative expression of a transgene in various plant tissues, thatmay not correspond to the patterns expected from transcriptionalregulatory elements present in the introduced gene construct. It is alsoobserved that the transgene insertion can affect the endogenous geneexpression. For these reasons, it is common to produce hundreds tothousands of different events and screen those events for a single eventthat has desired transgene expression levels and patterns for commercialpurposes. An event that has desired levels or patterns of transgeneexpression is useful for introgressing the transgene into other geneticbackgrounds by sexual outcrossing using conventional breeding methods.Progeny of such crosses maintain the transgene expressioncharacteristics of the original transformant. This strategy is used toensure reliable gene expression in a number of varieties that are welladapted to local growing conditions.

It would be advantageous to be able to detect the presence of aparticular event in order to determine whether progeny of a sexual crosscontain a transgene of interest. In addition, a method for detecting aparticular event would be helpful for complying with regulationsrequiring the pre-market approval and labeling of foods derived fromrecombinant crop plants or for use in environmental monitoring,monitoring traits in crops in the field or monitoring products derivedfrom a crop harvest, as well as for use in ensuring compliance ofparties subject to regulatory or contractual terms.

In the commercial production of crops, it is desirable to easily andquickly eliminate unwanted plants (i.e., “weeds”) from a field of cropplants. An ideal treatment would be one which could be applied to anentire field but which would eliminate only the unwanted plants whileleaving the crop plants unharmed. One such treatment system wouldinvolve the use of crop plants which are tolerant to an herbicide sothat when the herbicide was sprayed on a field of herbicide-tolerantcrop plants, the crop plants would continue to thrive whilenon-herbicide-tolerant weeds were killed or severely damaged.

Due to local and regional variation in dominant weed species as well aspreferred crop species, a continuing need exists for customized systemsof crop protection and weed management which can be adapted to the needsof a particular region, geography, and/or locality. Method andcompositions that allow for the rapid identification of events in plantsthat produce such qualities are needed. For example, a continuing needexists for methods of crop protection and weed management which canreduce the number of herbicide applications necessary to control weedsin a field, reduce the amount of herbicide necessary to control weeds ina field, reduce the amount of tilling necessary to produce a crop,and/or delay or prevent the development and/or appearance ofherbicide-resistant weeds. A continuing need exists for methods andcompositions of crop protection and weed management which allow thetargeted use of a particular herbicide and for the efficient detectionof such an event.

BRIEF SUMMARY OF THE INVENTION

Compositions and methods related to transgenic glyphosate-tolerantBrassica plants are provided. Specifically, the present inventionprovides Brassica plants containing a transgene which imparts toleranceto glyphosate. The event may be, for example, DP-073496-4. The Brassicaplant harboring the transgene at the recited chromosomal locationcomprises unique genomic/transgene junctions having at least thepolynucleotide sequence of SEQ ID NO: 2 or at least the polynucleotidesequence of SEQ ID NO: 12 and/or 13. Further provided are the seedsdeposited as Patent Deposit Number PTA-11504 and plants, plant cells,plant parts, seed and plant products derived therefrom. Characterizationof the genomic insertion site of DP-073496-4 or any other eventcomprising integration of the glyphosate-tolerance transgene providesfor an enhanced breeding efficiency and enables the use of molecularmarkers to track the transgene insert in the breeding populations andprogeny thereof. Various methods and compositions for theidentification, detection, and use of the glyphosate-N-acetyltransferase(“GAT” or “glyat”) transformation event in Brassica are provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows synthesis of plasmid PHP28181. Plasmid PHP28181 was used toproduce the GAT Brassica lines.

FIG. 2 provides a schematic map of plasmid PHP28181.

FIG. 3 provides a schematic map of insertion DNA, fragment PHP28181A.

FIG. 4 provides a schematic representation of fragment A from PHP28181(PHP28181A), specifically a schematic map of Hind III/Not I fragment(PHP28181A) containing the gat4621 gene cassette that was used for planttransformation to generate DP-073496-4 Brassica. The fragment size is2112 bp. The construct-specific primer locations of 09-0-3290/09-0-3288are indicated on the map.

FIG. 5 Southern analysis of Construct Specific PCR of Leaf DNA FromDP-073496-4 Brassica and Non-Genetically Modified Control Brassica. PCRamplification with primer set 09-0-3290/09-0-3288 targeting the uniqueubiquitin promoter and gat4621 junction present in DP-073496-4-canola.Expected PCR amplicon size is 675 bp.

FIG. 6 Southern analysis of Brassica FatA gene PCR of leaf DNA fromDP-073496-4 Brassica and Non-Genetically Modified Control Brassica. PCRamplification of endogenous brassica FatA gene with primer set09-0-2812/09-02813 as positive control for PCR amplification. ExpectedPCR amplicon size is 506 bp.

DETAILED DESCRIPTION OF THE INVENTION

The present inventions now will be described more fully hereinafter withreference to the accompanying drawings, in which some, but not allembodiments of the inventions are shown. Indeed, these inventions may beembodied in many different forms and should not be construed as limitedto the embodiments set forth herein; rather, these embodiments areprovided so that this disclosure will satisfy applicable legalrequirements. Like numbers refer to like elements throughout.

Many modifications and other embodiments of the inventions set forthherein will come to mind to one skilled in the art to which theseinventions pertain having the benefit of the teachings presented in theforegoing descriptions and the associated drawings. Therefore, it is tobe understood that the inventions are not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

Compositions and methods related to transgenic glyphosate-tolerantBrassica plants are provided. Specifically, the present inventionprovides Brassica plants having event DP-073496-4 or another eventcomprising PHP28181A or an operable fragment or variant thereof. ABrassica plant having event DP-073496-4, for example, has been modifiedby the insertion of the glyphosate acetyltransferase (glyat4621) genederived from Bacillus licheniformis. The glyat4621 gene was functionallyimproved by a gene shuffling process to optimize the kinetics ofglyphosate acetyltransferase (GLYAT) activity for acetylating theherbicide glyphosate. The insertion of the glyat4621 gene in the plantconfers tolerance to the herbicidal active ingredient glyphosate throughthe conversion of glyphosate to the non-toxic acetylated form. Thus, aBrassica plant having the event DP-073496-4 is tolerant to glyphosate.

The polynucleotides conferring the glyphosate tolerance are inserted ata specific position in the Brassica genome and thereby produce, forexample, the DP-073496-4 event. A Brassica plant harboring theDP-073496-4 event at a specific chromosomal location comprisesgenomic/transgene junctions having a unique polynucleotide sequenceexemplified by SEQ ID NO: 2 or at least the polynucleotide sequence ofSEQ ID NO: 12 and/or 13; SEQ ID NO: 14 and/or 15; or SEQ ID NO: 16and/or 17. The characterization of the genomic insertion site of eitherevent provides for an enhanced breeding efficiency and enables the useof molecular markers to track the transgene insert in the breedingpopulations and progeny thereof. Various methods and compositions forthe identification, detection, and use of the Brassica DP-073496-4 eventare provided herein. In one embodiment, a brassica plant having in itsgenome in the following order: a polynucleotide comprising SEQ ID NO:12, a polynucleotide encoding a glyphosate-N-acetyltransferase and apolynucleotide comprising SEQ ID NO: 13 is provided. The term “eventDP-073496-4 specific” refers to a polynucleotide sequence which issuitable for discriminatively identifying event DP-073496-4 in plants,plant material, or in products such as, but not limited to, oil producedfrom the seeds, or food or feed products (fresh or processed)comprising, or derived from, plant material.

Compositions further include seed deposited as Patent Deposit NumbersPTA-11504 and plants, plant cells, and seed derived therefrom.Applicant(s) have made a deposit of at least 2500 seeds of Brassicaevent DP-073496-4 with the American Type Culture Collection (ATCC),Manassas, Va. 20110-2209 USA on Nov. 24, 2010 and the deposit will bemaintained under the terms of the Budapest Treaty on the InternationalRecognition of the Deposit of Microorganisms for the Purposes of PatentProcedure. Deposits are made merely as a convenience for those of skillin the art and are not an admission that a deposit is required under 35U.S.C. §112. The seeds deposited with the ATCC are taken from thedeposit maintained by Pioneer Hi-Bred International, Inc., 7250 NW62^(nd) Avenue, Johnston, Iowa 50131-1000. Access to this deposit willbe available during the pendency of the application to the Commissionerof Patents and Trademarks and persons determined by the Commissioner tobe entitled thereto upon request. Upon allowance of any claims in theapplication, the Applicant(s) will make available to the public,pursuant to 37 C.F.R. §1.808, sample(s) of the deposit. The deposit ofseed comprising Brassica event DP-073496-4 will be maintained in theATCC depository, which is a public depository, for a period of 30 yearsor 5 years after the most recent request or for the enforceable life ofthe patent, whichever is longer and will be replaced if it becomesnonviable during that period. Additionally, Applicant(s) will havesatisfied all the requirements of 37 C.F.R. §§1.801-1.809, includingproviding an indication of the viability of the sample upon deposit.Applicant(s) have no authority to waive any restrictions imposed by lawon the transfer of biological material or its transportation incommerce. Applicant(s) do not waive any infringement of their rightsgranted under this patent or rights applicable to event DP-073496-4under the Plant Variety Protection Act (7 USC §2321, et seq.).Unauthorized seed multiplication prohibited. The seed may be regulated.

As used herein, the term “Brassica” means any Brassica plant andincludes all plant varieties that can be bred with Brassica. As usedherein, the term plant includes plant cells, plant organs, plantprotoplasts, plant cell tissue cultures from which plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants such as embryos, pollen, ovules, seeds,leaves, flowers, branches, fruit, stalks, roots, root tips, anthers, andthe like. Mature seed produced may be used for food, feed, fuel or othercommercial or industrial purposes or for purposes of growing orreproducing the species. Progeny, variants and mutants of theregenerated plants are also included within the scope of the invention,provided that these parts comprise a DP-073496-4 event.

A transgenic “event” is produced by transformation of plant cells with aheterologous DNA construct(s) including a nucleic acid expressioncassette that comprises a transgene of interest, the regeneration of apopulation of plants from cells which each comprise the insertedtransgene and selection of a particular plant characterized by insertioninto a particular genome location. An event is characterizedphenotypically by the expression of the transgene(s). At the geneticlevel, an event is part of the genetic makeup of a plant. The term“event” also refers to progeny, produced by a sexual outcross betweenthe transformant and another variety, that include the heterologous DNA.Even after repeated back-crossing to a recurrent parent, the insertedDNA and flanking DNA from the transformed parent are present in theprogeny of the cross at the same chromosomal location. The term “event”also refers to DNA from the original transformant comprising theinserted DNA and flanking sequence immediately adjacent to the insertedDNA that would be expected to be transferred to a progeny as the resultof a sexual cross of one parental line that includes the inserted DNA(e.g., the original transformant and progeny resulting from selfing) anda parental line that does not contain the inserted DNA.

As used herein, “insert DNA” refers to the heterologous DNA within theexpression cassettes used to transform the plant material while“flanking DNA” can comprise either genomic DNA naturally present in anorganism such as a plant, or foreign (heterologous) DNA introduced viathe transformation process which is extraneous to the original insertDNA molecule, e.g. fragments associated with the transformation event. A“flanking region” or “flanking sequence” as used herein refers to asequence of at least 20, 50, 100, 200, 300, 400, 1000, 1500, 2000, 2500or 5000 base pairs or greater which is located either immediatelyupstream of and contiguous with, or immediately downstream of andcontiguous with, the original foreign insert DNA molecule. Non-limitingexamples of the flanking regions of the DP-073496-4 event comprisepolynucleotide sequences that are set forth in SEQ ID NO: 2, 8 and/or 9and variants and fragments thereof.

Transformation procedures leading to random integration of the foreignDNA will result in transformants containing different flanking regionscharacteristic of and unique for each transformant. When recombinant DNAis introduced into a plant through traditional crossing, its flankingregions will generally not be changed. Transformants will also containunique junctions between a piece of heterologous insert DNA and genomicDNA or two pieces of genomic DNA or two pieces of heterologous DNA. A“junction” is a point where two specific DNA fragments join. Forexample, a junction exists where insert DNA joins flanking DNA. Ajunction point also exists in a transformed organism where two DNAfragments join together in a manner that is modified from that found inthe native organism. As used herein, “junction DNA” refers to DNA thatcomprises a junction point. Non-limiting examples of junction DNA fromthe DP-073496-4 event are forth in SEQ ID NO: 2, 11, 12, 13, 14, 15, 16,17, 18, and/or 19 or variants and fragments thereof.

A DP073496-4 plant can be bred by first sexually crossing a firstparental Brassica plant grown from the transgenic DP-073496-4 Brassicaplant (or progeny thereof derived from transformation with theexpression cassettes of the embodiments of the present invention thatconfer herbicide tolerance) and a second parental Brassica plant thatlacks the herbicide tolerance phenotype, thereby producing a pluralityof first progeny plants and then selecting a first progeny plant thatdisplays the desired herbicide tolerance and selfing the first progenyplant, thereby producing a plurality of second progeny plants and thenselecting from the second progeny plants which display the desiredherbicide tolerance. These steps can further include the back-crossingof the first herbicide tolerant progeny plant or the second herbicidetolerant progeny plant to the second parental Brassica plant or a thirdparental Brassica plant, thereby producing a Brassica plant thatdisplays the desired herbicide tolerance. It is further recognized thatassaying progeny for phenotype is not required. Various methods andcompositions, as disclosed elsewhere herein, can be used to detectand/or identify the DP073496-4 or other event.

Two different transgenic plants can also be sexually crossed to produceoffspring that contain two independently-segregating exogenous genes.Selfing of appropriate progeny can produce plants that are homozygousfor both exogenous genes. Back-crossing to a parental plant andout-crossing with a non-transgenic plant are also contemplated, as isvegetative propagation. Descriptions of other breeding methods that arecommonly used for different traits and crops can be found in one ofseveral references, e.g., Fehr, in Breeding Methods for CultivarDevelopment, Wilcos, ed., American Society of Agronomy, Madison Wis.(1987).

The term “germplasm” refers to an individual, a group of individuals ora clone representing a genotype, variety, species or culture or thegenetic material thereof.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generally isogenicor near isogenic.

Inbred lines tend to be highly homogeneous, homozygous and reproducible.Many analytical methods are available to determine the homozygosity andphenotypic stability of inbred lines.

The phrase “hybrid plants” refers to plants which result from a crossbetween genetically different individuals.

The term “crossed” or “cross” in the context of this invention means thefusion of gametes, e.g., via pollination to produce progeny (i.e.,cells, seeds, or plants) in the case of plants. The term encompassesboth sexual crosses (the pollination of one plant by another) and, inthe case of plants, selfing (self-pollination, i.e., when the pollen andovule are from the same plant).

The term “introgression” refers to the transmission of a desired alleleof a genetic locus from one genetic background to another. In onemethod, the desired alleles can be introgressed through a sexual crossbetween two parents, wherein at least one of the parents has the desiredallele in its genome.

In some embodiments, the polynucleotides conferring the brassicaDP-073496-4 event of the invention are engineered into a molecularstack. In other embodiments, the molecular stack further comprises atleast one additional polynucleotide that confers tolerance to a secondherbicide. In one embodiment, the sequence confers tolerance toglufosinate, and in a specific embodiment, the sequence comprises patgene. In another embodiment, the additional polynucleotide providestolerance to ALS-inhibitor herbicides.

In other embodiments, an event of the invention comprises one or moretraits of interest, and in more specific embodiments, the plant isstacked with any combination of polynucleotide sequences of interest inorder to create plants with a desired combination of traits. A trait, asused herein, refers to the phenotype derived from a particular sequenceor groups of sequences. For example, herbicide-tolerance polynucleotidesmay be stacked with any other polynucleotides encoding polypeptideshaving pesticidal and/or insecticidal activity, such as Bacillusthuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892;5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser, et al., (1986) Gene48:109; Lee, et al., (2003) Appl. Environ. Microbiol. 69:4648-4657(Vip3A); Galitzky, et al., (2001) Acta Crystallogr. D. Biol.Crystallogr. 57:1101-1109 (Cry3Bb1) and Herman, et al., (2004) J. Agric.Food Chem. 52:2726-2734 (Cry1F)), lectins (Van Damme, et al., (1994)Plant Mol. Biol. 24:825, pentin (described in U.S. Pat. No. 5,981,722),and the like. The combinations generated can also include multiplecopies of any one of the polynucleotides of interest.

In some embodiments, an event of the invention may be stacked with otherherbicide-tolerance traits to create a transgenic plant of the inventionwith further improved properties. Other herbicide-tolerancepolynucleotides that could be used in such embodiments include thoseconferring tolerance to glyphosate by other modes of action, such as,for example, a gene that encodes a glyphosate oxido-reductase enzyme asdescribed more fully in U.S. Pat. Nos. 5,776,760 and 5,463,175. Othertraits that could be combined with an event of the invention includethose derived from polynucleotides that confer on the plant the capacityto produce a higher level of 5-enolpyruvylshikimate-3-phosphate synthase(EPSPS), for example, as more fully described in U.S. Pat. Nos.6,248,876 B1; 5,627,061; 5,804,425; 5,633,435; 5,145,783; 4,971,908;5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366;5,310,667; 4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE37,287 E and 5,491,288 and International Publication Numbers WO97/04103; WO 00/66746; WO 01/66704 and WO 00/66747. Other traits thatcould be combined with the an event of the invention include thoseconferring tolerance to sulfonylurea and/or imidazolinone, for example,as described more fully in U.S. Pat. Nos. 5,605,011; 5,013,659;5,141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107;5,928,937 and 5,378,824 and International Publication Number WO96/33270.

In some embodiments, an event of the invention may be stacked with, forexample, hydroxyphenylpyruvatedioxygenases which are enzymes thatcatalyze the reaction in which para-hydroxyphenylpyruvate (HPP) istransformed into homogentisate. Molecules which inhibit this enzyme andwhich bind to the enzyme in order to inhibit transformation of the HPPinto homogentisate are useful as herbicides. Traits conferring toleranceto such herbicides in plants are described in U.S. Pat. Nos. 6,245,968B1; 6,268,549 and 6,069,115 and International Publication Number WO99/23886. Other examples of suitable herbicide-tolerance traits thatcould be stacked with an event of the invention include aryloxyalkanoatedioxygenase polynucleotides (which reportedly confer tolerance to 2,4-Dand other phenoxy auxin herbicides as well as toaryloxyphenoxypropionate herbicides as described, for example, inInternational Publication WO 05/107437) and dicamba-tolerancepolynucleotides as described, for example, in Herman, et al., (2005) J.Biol. Chem. 280:24759-24767.

Other examples of herbicide-tolerance traits that could be combined withan event disclosed herein include those conferred by polynucleotidesencoding an exogenous phosphinothricin acetyltransferase, as describedin U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675;5,561,236; 5,648,477; 5,646,024; 6,177,616 and 5,879,903. Plantscontaining an exogenous phosphinothricin acetyltransferase can exhibitimproved tolerance to glufosinate herbicides, which inhibit the enzymeglutamine synthase. Other examples of herbicide-tolerance traits thatcould be combined with an event disclosed herein include those conferredby polynucleotides conferring altered protoporphyrinogen oxidase(protox) activity, as described in U.S. Pat. Nos. 6,288,306 B1;6,282,837 B1 and 5,767,373 and International Publication Number WO01/12825. Plants containing such polynucleotides can exhibit improvedtolerance to any of a variety of herbicides which target the protoxenzyme (also referred to as “protox inhibitors”).

In other embodiments, an ALS inhibitor-tolerant trait is combined withthe event disclosed herein. As used herein, an “ALS inhibitor-tolerantpolypeptide” comprises any polypeptide which when expressed in a plantconfers tolerance to at least one ALS inhibitor. A variety of ALSinhibitors are known and include, for example, sulfonylurea,imidazolinone, triazolopyrimidines, pryimidinyoxy(thio)benzoates, and/orsulfonylaminocarbonyltriazolinone herbicide. Additional ALS inhibitorsare known and are disclosed elsewhere herein. It is known in the artthat ALS mutations fall into different classes with regard to toleranceto sulfonylureas, imidazolinones, triazolopyrimidines, andpyrimidinyl(thio)benzoates, including mutations having the followingcharacteristics: (1) broad tolerance to all four of these groups; (2)tolerance to imidazolinones and pyrimidinyl(thio)benzoates; (3)tolerance to sulfonylureas and triazolopyrimidines; and (4) tolerance tosulfonylureas and imidazolinones.

Various ALS inhibitor-tolerant polypeptides can be employed. In someembodiments, the ALS inhibitor-tolerant polynucleotides contain at leastone nucleotide mutation resulting in one amino acid change in the ALSpolypeptide. In specific embodiments, the change occurs in one of sevensubstantially conserved regions of acetolactate synthase. See, forexample, Hattori et al. (1995) Molecular Genetics and Genomes246:419-425; Lee et al. (1998) EMBO Journal 7:1241-1248; Mazur et al.(1989) Ann. Rev. Plant Phys. 40:441-470; and U.S. Pat. No. 5,605,011,each of which is incorporated by reference in their entirety. The ALSinhibitor-tolerant polypeptide can be encoded by, for example, the SuRAor SuRB locus of ALS. In specific embodiments, the ALSinhibitor-tolerant polypeptide comprises the C3 ALS mutant, the HRA ALSmutant, the S4 mutant or the S4/HRA mutant or any combination thereof.Different mutations in ALS are known to confer tolerance to differentherbicides and groups (and/or subgroups) of herbicides; see, e.g.,Tranel and Wright (2002) Weed Science 50:700-712. See also, U.S. Pat.Nos. 5,605,011, 5,378,824, 5,141,870, and 5,013,659, each of which isherein incorporated by reference in their entirety. See also, SEQ IDNO:65 comprising a soybean HRA sequence; SEQ ID NO:66 comprising a maizeHRA sequence; SEQ ID NO:67 comprising an Arabidopsis HRA sequence; andSEQ ID NO:86 comprising an HRA sequence used in cotton. The HRA mutationin ALS finds particular use in one embodiment of the invention. Themutation results in the production of an acetolactate synthasepolypeptide which is resistant to at least one ALS inhibitor chemistryin comparison to the wild-type protein. For example, a plant expressingan ALS inhibitor-tolerant polypeptide may be tolerant of a dose ofsulfonylurea, imidazolinone, triazolopyrimidines,pryimidinyloxy(thio)benzoates, and/or sulfonylaminocarbonyltriazolinoneherbicide that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30,35, 40, 50, 70, 80, 100, 125, 150, 200, 500, or 1000 times higher than adose of the herbicide that would cause damage to an appropriate controlplant. In some embodiments, an ALS inhibitor-tolerant polypeptidecomprises a number of mutations. Additionally, plants having an ALSinhibitor polypeptide can be generated through the selection ofnaturally occurring mutations that impart tolerance to glyphosate.

In some embodiments, the ALS inhibitor-tolerant polypeptide conferstolerance to sulfonylurea and imidazolinone herbicides. Sulfonylurea andimidazolinone herbicides inhibit growth of higher plants by blockingacetolactate synthase (ALS), also known as, acetohydroxy acid synthase(AHAS). For example, plants containing particular mutations in ALS(e.g., the S4 and/or HRA mutations) are tolerant to sulfonylureaherbicides. The production of sulfonylurea-tolerant plants andimidazolinone-tolerant plants is described more fully in U.S. Pat. Nos.5,605,011; 5,013,659; 5,141,870; 5,767,361; 5,731,180; 5,304,732;4,761,373; 5,331,107; 5,928,937; and 5,378,824; and internationalpublication WO 96/33270, which are incorporated herein by reference intheir entireties for all purposes. In specific embodiments, the ALSinhibitor-tolerant polypeptide comprises a sulfonamide-tolerantacetolactate synthase (otherwise known as a sulfonamide-tolerantacetohydroxy acid synthase) or an imidazolinone-tolerant acetolactatesynthase (otherwise known as an imidazolinone-tolerant acetohydroxy acidsynthase).

Other examples of herbicide-tolerance traits that could be combined withan event disclosed herein include those conferring tolerance to at leastone herbicide in a plant such as, for example, a brassica plant orhorseweed. Herbicide-tolerant weeds are known in the art, as are plantsthat vary in their tolerance to particular herbicides. See, e.g., Greenand Williams, (2004) “Correlation of Corn (Zea mays) Inbred Response toNicosulfuron and Mesotrione,” poster presented at the WSSA AnnualMeeting in Kansas City, Mo., Feb. 9-12, 2004; Green, (1998) WeedTechnology 12:474-477; Green and Ulrich, (1993) Weed Science 41:508-516.The trait(s) responsible for these tolerances can be combined bybreeding or via other methods with an event disclosed herein to providea plant of the invention as well as methods of use thereof.

An event disclosed herein can also be combined with at least one othertrait to produce plants of the present invention that further comprise avariety of desired trait combinations including, but not limited to,traits desirable for animal feed such as high oil content (e.g., U.S.Pat. No. 6,232,529); balanced amino acid content (e.g., hordothionins(U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802 and 5,703,409; U.S. Pat.No. 5,850,016); barley high lysine (Williamson, et al., (1987) Eur. J.Biochem. 165:99-106 and WO 98/20122) and high methionine proteins(Pedersen, et al., (1986) J. Biol. Chem. 261:6279; Kirihara, et al.,(1988) Gene 71:359 and Musumura, et al., (1989) Plant Mol. Biol.12:123)); increased digestibility (e.g., modified storage proteins (U.S.patent application Ser. No. 10/053,410, filed Nov. 7, 2001) andthioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3,2001)); the disclosures of which are herein incorporated by reference.Desired trait combinations also include LLNC (low linolenic acidcontent; see, e.g., Dyer, et al., (2002) Appl. Microbiol. Biotechnol.59:224-230) and OLCH (high oleic acid content; see, e.g.,Fernandez-Moya, et al., (2005) J. Agric. Food Chem. 53:5326-5330).

An event disclosed herein may also be combined with other desirabletraits such as, for example, fumonisin detoxification genes (U.S. Pat.No. 5,792,931), avirulence and disease resistance genes (Jones, et al.,(1994) Science 266:789; Martin, et al., (1993) Science 262:1432;Mindrinos, et al., (1994) Cell 78:1089) and traits desirable forprocessing or process products such as modified oils (e.g., fatty aciddesaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modifiedstarches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS),starch branching enzymes (SBE), and starch debranching enzymes (SDBE))and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321;beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoAreductase (Schubert, et al., (1988) J. Bacteriol. 170:5837-5847)facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosuresof which are herein incorporated by reference. One could also combineherbicide-tolerant polynucleotides with polynucleotides providingagronomic traits such as male sterility (e.g., see, U.S. Pat. No.5,583,210), stalk strength, flowering time or transformation technologytraits such as cell cycle regulation or gene targeting (e.g., WO99/61619, WO 00/17364 and WO 99/25821), the disclosures of which areherein incorporated by reference.

In another embodiment, an event disclosed herein can also be combinedwith the Rcg1 sequence or biologically active variant or fragmentthereof. The Rcg1 sequence is an anthracnose stalk rot resistance genein corn. See, for example, U.S. patent application Ser. Nos. 11/397,153,11/397,275 and 11/397,247, each of which is herein incorporated byreference.

These stacked combinations can be created by any method including, butnot limited to, breeding plants by any conventional methodology orgenetic transformation. If the sequences are stacked by geneticallytransforming the plants, the polynucleotide sequences of interest can becombined at any time and in any order. The traits can be introducedsimultaneously in a co-transformation protocol with the polynucleotidesof interest provided by any combination of transformation cassettes. Forexample, if two sequences will be introduced, the two sequences can becontained in separate transformation cassettes (trans) or contained onthe same transformation cassette (cis). Expression of the sequences canbe driven by the same promoter or by different promoters. In certaincases, it may be desirable to introduce a transformation cassette thatwill suppress the expression of the polynucleotide of interest. This maybe combined with any combination of other suppression cassettes oroverexpression cassettes to generate the desired combination of traitsin the plant. It is further recognized that polynucleotide sequences canbe stacked at a desired genomic location using a site-specificrecombination system. See, for example, WO99/25821, WO99/25854,WO99/25840, WO99/25855 and WO99/25853, all of which are hereinincorporated by reference.

As used herein, the use of the term “polynucleotide” is not intended tolimit the present invention to polynucleotides comprising DNA. Those ofordinary skill in the art will recognize that polynucleotides, cancomprise ribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides. Such deoxyribonucleotides and ribonucleotidesinclude both naturally occurring molecules and synthetic analogues. Thepolynucleotides of the invention also encompass all forms of sequencesincluding, but not limited to, single-stranded forms, double-strandedforms, hairpins, stem-and-loop structures, and the like.

A DP-073496-4 Brassica plant comprises an expression cassette having anoptimized glyphosate acetyltransferase polynucleotide. The cassette caninclude 5′ and 3′ regulatory sequences operably linked to the glyatpolynucleotides. “Operably linked” is intended to mean a functionallinkage between two or more elements. For example, an operable linkagebetween a polynucleotide of interest and a regulatory sequence (i.e., apromoter) is functional link that allows for the expression of thepolynucleotide of interest. Operably linked elements may be contiguousor non-contiguous. When used to refer to the joining of two proteincoding regions, by operably linked it is intended that the codingregions are in the same reading frame. The cassette may additionallycontain at least one additional gene to be cotransformed into theorganism. Alternatively, the additional gene(s) can be provided onmultiple expression cassettes. Such an expression cassette is providedwith a plurality of restriction sites and/or recombination sites forinsertion of the polynucleotide to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain selectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a coding region and a transcriptional andtranslational termination region functional in plants. “Promoter” refersto a nucleotide sequence capable of controlling the expression of acoding sequence or functional RNA. In general, a coding sequence islocated 3′ to a promoter sequence. The promoter sequence can compriseproximal and more distal upstream elements, the latter elements areoften referred to as enhancers. Accordingly, an “enhancer” is anucleotide sequence that can stimulate promoter activity and may be aninnate element of the promoter or a heterologous element inserted toenhance the level or tissue-specificity of a promoter. Promoters may bederived in their entirety from a native gene, or be composed ofdifferent elements derived from different promoters found in nature oreven comprise synthetic nucleotide segments. It is understood by thoseskilled in the art that different promoters may direct the expression ofa gene in different tissues or cell types or at different stages ofdevelopment or in response to different environmental conditions.Promoters that cause a nucleic acid fragment to be expressed in mostcell types at most times are commonly referred to as “constitutivepromoters”. New promoters of various types useful in plant cells areconstantly being discovered; numerous examples may be found in thecompilation by Okamuro and Goldberg, (1989) Biochemistry of Plants15:1-82. It is further recognized that since in most cases the exactboundaries of regulatory sequences have not been completely defined,nucleic acid fragments of different lengths may have identical promoteractivity.

The expression cassettes may also contain 5′ leader sequences. Suchleader sequences can act to enhance translation. The regulatory regions(i.e., promoters, transcriptional regulatory regions, RNA processing orstability regions, introns, polyadenylation signals, transcriptionaltermination regions and translational termination regions) and/or thecoding region may be native/analogous or heterologous to the host cellor to each other.

The “translation leader sequence” refers to a nucleotide sequencelocated between the promoter sequence of a gene and the coding sequence.The translation leader sequence is present in the fully processed mRNAupstream of the translation start sequence. The translation leadersequence may affect numerous parameters including, processing of theprimary transcript to mRNA, mRNA stability and/or translationefficiency. Examples of translation leader sequences have been described(Turner and Foster, (1995) Mol. Biotechnol. 3:225-236). The “3′non-coding sequences” refer to nucleotide sequences located downstreamof a coding sequence and include polyadenylation recognition sequencesand other sequences encoding regulatory signals capable of affectingmRNA processing or gene expression. The polyadenylation signal isusually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht, et al., (1989) PlantCell 1:671-680.

As used herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus or the promoteris not the native promoter for the operably linked polynucleotide.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved. The expression cassette can alsocomprise a selectable marker gene for the selection of transformedcells. Selectable marker genes are utilized for the selection oftransformed cells or tissues.

Isolated polynucleotides are provided that can be used in variousmethods for the detection and/or identification of the brassicaDP-073496-4 event. An “isolated” or “purified” polynucleotide orbiologically active portion thereof, is substantially or essentiallyfree from components that normally accompany or interact with thepolynucleotide as found in its naturally occurring environment. Thus, anisolated or purified polynucleotide is substantially free of othercellular material or culture medium when produced by recombinanttechniques or substantially free of chemical precursors or otherchemicals when chemically synthesized. Optimally, an “isolated”polynucleotide is free of sequences (optimally protein encodingsequences) that naturally flank the polynucleotide (i.e., sequenceslocated at the 5′ and 3′ ends of the polynucleotide) in the genomic DNAof the organism from which the polynucleotide is derived. For example,in various embodiments, the isolated polynucleotide can contain lessthan about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotidesequence that naturally flank the polynucleotide in genomic DNA of thecell from which the polynucleotide is derived.

In specific embodiments, the polynucleotides of the invention comprisethe junction DNA sequence set forth in NO: 2, or variants and/orfragments thereof or the junction DNA sequence set forth in SEQ ID NO:12and/or 13. In other embodiments, the polynucleotides of the inventioncomprise the junction DNA sequences set forth in SEQ ID NO: 14, 15, 16,17, 18 and/or 19 or variants and fragments thereof. In specificembodiments, methods of detection described herein amplify apolynucleotide comprising a junction of the specific DP-073496-4 event.Fragments and variants of junction DNA sequences are suitable fordiscriminatively identifying either event DP-073496-4. As discussedelsewhere herein, such sequences find use as primers and/or probes.

In other embodiments, the polynucleotides of the invention comprisepolynucleotides that can detect a DP-073496-4 event or a region specificto DP-073496-4. Such sequences include any polynucleotide set forth inSEQ ID NO: 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27 or variants and fragments thereof.Fragments and variants of polynucleotides that detect a DP-073496-4event or a region specific to DP-073496-4 are suitable fordiscriminatively identifying event DP-073496-4. As discussed elsewhereherein, such sequences find use as primers and/or probes.

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a polynucleotide having deletions(i.e., truncations) at the 5′ and/or 3′ end; deletion and/or addition ofone or more nucleotides at one or more internal sites in the nativepolynucleotide and/or substitution of one or more nucleotides at one ormore sites in the native polynucleotide.

As used herein, a “probe” is an isolated polynucleotide to which isattached a conventional detectable label or reporter molecule, e.g., aradioactive isotope, ligand, chemiluminescent agent, enzyme, etc. Such aprobe is complementary to a strand of a target polynucleotide. In thecase of the present invention, the probe is complementary to a strand ofisolated DNA from Brassica event DP-073496-4, whether from a Brassicaplant or from a sample that includes DNA from the event. Probesaccording to the present invention include not only deoxyribonucleic orribonucleic acids but also polyamides and other probe materials that canspecifically detect the presence of the target DNA sequence.

As used herein, “primers” are isolated polynucleotides that are annealedto a complementary target DNA strand by nucleic acid hybridization toform a hybrid between the primer and the target DNA strand, thenextended along the target DNA strand by a polymerase, e.g., a DNApolymerase. Primer pairs of the invention refer to their use foramplification of a target polynucleotide, e.g., by the polymerase chainreaction (PCR) or other conventional nucleic-acid amplification methods.“PCR” or “polymerase chain reaction” is a technique used for theamplification of specific DNA segments (see, U.S. Pat. Nos. 4,683,195and 4,800,159, herein incorporated by reference). Any combination ofprimers can be used such that the pair allows for the detection of aDP-073496-4 event or a region specific to DP-073496-4.

Probes and primers are of sufficient nucleotide length to bind to thetarget DNA sequence and specifically detect and/or identify apolynucleotide having a DP-073496-4 event. It is recognized that thehybridization conditions or reaction conditions can be determined by theoperator to achieve this result. This length may be of any length thatis of sufficient length to be useful in a detection method of choice.Generally, 8, 11, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50, 75, 100,200, 300, 400, 500, 600, 700 nucleotides or more or between about 11-20,20-30, 30-40, 40-50, 50-100, 100-200, 200-300, 300-400, 400-500,500-600, 600-700, 700-800 or more nucleotides in length are used. Suchprobes and primers can hybridize specifically to a target sequence underhigh stringency hybridization conditions. Probes and primers accordingto embodiments of the present invention may have complete DNA sequenceidentity of contiguous nucleotides with the target sequence, althoughprobes differing from the target DNA sequence and that retain theability to specifically detect and/or identify a target DNA sequence maybe designed by conventional methods. Accordingly, probes and primers canshare about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or greater sequence identity or complementarity to the targetpolynucleotide, or can differ from the target sequence by 1, 2, 3, 4, 5,6 or more nucleotides. Probes can be used as primers, but are generallydesigned to bind to the target DNA or RNA and are not used in anamplification process. In one non-limiting embodiment, a probe cancomprises a polynucleotide encoding the glyat4621 sequence or anyvariant or fragment thereof.

Specific primers can be used to amplify an integration fragment toproduce an amplicon that can be used as a “specific probe” or can itselfbe detected for identifying event DP-073496-4 in biological samples.Alternatively, a probe of the invention can be used during the PCRreaction to allow for the detection of the amplification event (i.e., aTaqman™ probe or an MGB probe, so called real-time PCR). When the probeis hybridized with the polynucleotides of a biological sample underconditions which allow for the binding of the probe to the sample, thisbinding can be detected and thus allow for an indication of the presenceof event DP-073496-4 in the biological sample. Such identification of abound probe has been described in the art. In an embodiment of theinvention, the specific probe is a sequence which, under optimizedconditions, hybridizes specifically to a region within the 5′ or 3′flanking region of the event and also comprises a part of the foreignDNA contiguous therewith. The specific probe may comprise a sequence ofat least 80%, between 80 and 85%, between 85 and 90%, between 90 and 95%and between 95 and 100% identical (or complementary) to a specificregion of the DP-073496-4 event.

As used herein, “amplified DNA” or “amplicon” refers to the product ofpolynucleotide amplification of a target polynucleotide that is part ofa nucleic acid template. For example, to determine whether a Brassicaplant resulting from a sexual cross contains the DP-073496-4 event, DNAextracted from the Brassica plant tissue sample may be subjected to apolynucleotide amplification method using a DNA primer pair thatincludes a first primer derived from flanking sequence adjacent to theinsertion site of inserted heterologous DNA and a second primer derivedfrom the inserted heterologous DNA to produce an amplicon that isdiagnostic for the presence of the DP-073496-4 event DNA. In specificembodiments, the amplicon comprises a DP-073496-4 junctionpolynucleotide (i.e., a portion of SEQ ID NO: 2 which spans the junctionsite, such as, for example, SEQ ID NO: 10, 11, 12, 13, 14, 15, 16, 17,18 and/or 19 or variants and fragments thereof). By “diagnostic” for aDP-073496-4 event, the use of any method or assay which discriminatesbetween the presence or the absence of a DP-073496-4 event in abiological sample is intended. Alternatively, the second primer may bederived from the flanking sequence. In still other embodiments, primerpairs can be derived from flanking sequence on both sides of theinserted DNA so as to produce an amplicon that includes the entireinsert polynucleotide of the expression construct as well as thesequence flanking the transgenic insert. See, FIG. 3. The amplicon is ofa length and has a sequence that is also diagnostic for the event (i.e.,has a junction DNA from a DP-073496-4 event). The amplicon may range inlength from the combined length of the primer pairs plus one nucleotidebase pair to any length of amplicon producible by a DNA amplificationprotocol. A member of a primer pair derived from the flanking sequencemay be located a distance from the inserted DNA sequence, this distancecan range from one nucleotide base pair up to the limits of theamplification reaction or about twenty thousand nucleotide base pairs.The use of the term “amplicon” specifically excludes primer dimers thatmay be formed in the DNA thermal amplification reaction.

Methods for preparing and using probes and primers are described, forexample, in Molecular Cloning: A Laboratory Manual, 2.sup.nd ed, vol.1-3, ed. Sambrook, et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. 1989 (hereinafter, “Sambrook, et al., 1989”);Current Protocols in Molecular Biology, ed. Ausubel, et al., GreenePublishing and Wiley-Interscience, New York, 1992 (with periodicupdates) (hereinafter, “Ausubel, et al., 1992”) and Innis, et al., PCRProtocols: A Guide to Methods and Applications, Academic Press: SanDiego, 1990. PCR primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asthe PCR primer analysis tool in Vector NTI version 6 (Informax Inc.,Bethesda Md.); PrimerSelect (DNASTAR Inc., Madison, Wis.); and Primer(Version 0.5.COPYRGT., 1991, Whitehead Institute for BiomedicalResearch, Cambridge, Mass.). Additionally, the sequence can be visuallyscanned and primers manually identified using guidelines known to one ofskill in the art.

It is to be understood that as used herein the term “transgenic”includes any cell, cell line, callus, tissue, plant part or plant, thegenotype of which has been altered by the presence of a heterologousnucleic acid including those transgenics initially so altered as well asthose created by sexual crosses or asexual propagation from the initialtransgenic. The term “transgenic” as used herein does not encompass thealteration of the genome (chromosomal or extra-chromosomal) byconventional plant breeding methods or by naturally occurring eventssuch as random cross-fertilization, non-recombinant viral infection,non-recombinant bacterial transformation, non-recombinant transpositionor spontaneous mutation.

“Transformation” refers to the transfer of a nucleic acid fragment intothe genome of a host organism, resulting in genetically stableinheritance. Host organisms containing the transformed nucleic acidfragments are referred to as “transgenic” organisms. Examples of methodsof plant transformation include Agrobacterium-mediated transformation(De Blaere, et al., (1987) Meth. Enzymol. 143:277) andparticle-accelerated or “gene gun” transformation technology (Klein, etal., (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050,incorporated herein by reference). Additional transformation methods aredisclosed below.

Thus, isolated polynucleotides of the invention can be incorporated intorecombinant constructs, typically DNA constructs, which are capable ofintroduction into and replication in a host cell. Such a construct canbe a vector that includes a replication system and sequences that arecapable of transcription and translation of a polypeptide-encodingsequence in a given host cell. A number of vectors suitable for stabletransfection of plant cells or for the establishment of transgenicplants have been described in, e.g., Pouwels, et al., (1985; Supp. 1987)Cloning Vectors: A Laboratory Manual, Weissbach and Weissbach, (1989)Methods for Plant Molecular Biology (Academic Press, New York) andFlevin, et al., (1990) Plant Molecular Biology Manual (Kluwer AcademicPublishers). Typically, plant expression vectors include, for example,one or more cloned plant genes under the transcriptional control of 5′and 3′ regulatory sequences and a dominant selectable marker. Such plantexpression vectors also can contain a promoter regulatory region (e.g.,a regulatory region controlling inducible or constitutive,environmentally- or developmentally-regulated or cell- ortissue-specific expression), a transcription initiation start site, aribosome binding site, an RNA processing signal, a transcriptiontermination site and/or a polyadenylation signal.

Various methods and compositions for identifying event DP-073496-4 areprovided. Such methods find use in identifying and/or detecting aDP-073496-4 event in any biological material. Such methods include, forexample, methods to confirm seed purity and methods for screening seedsin a seed lot for a DP-073496-4 event. In one embodiment, a method foridentifying event DP-073496-4 in a biological sample is provided andcomprises contacting the sample with a first and a second primer; and,amplifying a polynucleotide comprising a DP-073496-4 specific region.

A biological sample can comprise any sample in which one desires todetermine if DNA having event DP-073496-4 is present. For example, abiological sample can comprise any plant material or material comprisingor derived from a plant material such as, but not limited to, food orfeed products. As used herein, “plant material” refers to material whichis obtained or derived from a plant or plant part. In specificembodiments, the biological sample comprises a brassica tissue.

Primers and probes based on the flanking DNA and insert sequencesdisclosed herein can be used to confirm (and, if necessary, to correct)the disclosed sequences by conventional methods, e.g., by re-cloning andsequencing such sequences. The polynucleotide probes and primers of thepresent invention specifically detect a target DNA sequence. Anyconventional nucleic acid hybridization or amplification method can beused to identify the presence of DNA from a transgenic event in asample. By “specifically detect” it is intended that the polynucleotidecan be used either as a primer to amplify a DP-073496-4 specific regionor the polynucleotide can be used as a probe that hybridizes understringent conditions to a polynucleotide from a DP-073496-4 event. Thelevel or degree of hybridization which allows for the specific detectionof a DP-073496-4 event or a specific region of a DP-073496-4 event issufficient to distinguish the polynucleotide with the DP-073496-4specific region from a polynucleotide lacking this region and therebyallow for discriminately identifying a DP-073496-4 event. By “sharessufficient sequence identity or complentarity to allow for theamplification of a DP-073496-4 specific event” is intended the sequenceshares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity or complementarity to a fragment or across the fulllength of the polynucleotide from the DP-073496-4 specific region.

Regarding the amplification of a target polynucleotide (e.g., by PCR)using a particular amplification primer pair, “stringent conditions” areconditions that permit the primer pair to hybridize to the targetpolynucleotide to which one primer having the corresponding wild-typesequence (or its complement) and another primer having the correspondingDP-073496-4 inserted DNA sequence would bind and preferably to producean identifiable amplification product (the amplicon) having aDP-073496-4 specific region in a DNA thermal amplification reaction. Ina PCR approach, oligonucleotide primers can be designed for use in PCRreactions to amplify a DP-073496-4 specific region. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). See also, Innis, et al., eds. (1990) PCR Protocols: AGuide to Methods and Applications (Academic Press, New York); Innis andGelfand, eds. (1995) PCR Strategies (Academic Press, New York) and Innisand Gelfand, eds. (1999) PCR Methods Manual (Academic Press, New York).Methods of amplification are further described in U.S. Pat. Nos.4,683,195, 4,683,202 and Chen, et al., (1994) PNAS 91:5695-5699. Thesemethods as well as other methods known in the art of DNA amplificationmay be used in the practice of the embodiments of the present invention.It is understood that a number of parameters in a specific PCR protocolmay need to be adjusted to specific laboratory conditions and may beslightly modified and yet allow for the collection of similar results.These adjustments will be apparent to a person skilled in the art.

The amplified polynucleotide (amplicon) can be of any length that allowsfor the detection of the DP-073496-4 event or a DP-073496-4 specificregion. For example, the amplicon can be about 10, 50, 100, 200, 300,500, 700, 100, 2000, 3000, 4000, 5000 nucleotides in length or longer.

In specific embodiments, the specific region of the DP-073496-4 event isdetected.

Any primer can be employed in the methods of the invention that allows aDP-073496-4 specific region to be amplified and/or detected. Forexample, in specific embodiments, the first primer comprises a fragmentof a polynucleotide of SEQ ID NO: 2 or 3, wherein the first or thesecond primer shares sufficient sequence identity or complementarity tothe polynucleotide to amplify the DP-073496-4 specific region. Theprimer pair can comprise a fragment of SEQ ID NO: 2 or 3. In anotherembodiment, the primer pair comprises a first primer comprising afragment of SEQ ID NO: 8 and a second primer comprising a fragment ofSEQ ID NO: 9 or 10; or, alternatively, the primer pair comprises a firstprimer comprising a fragment of SEQ ID NO: 9 and the second primercomprises a fragment of SEQ ID NO: 8 or 10. The primers can be of anylength sufficient to amplify a DP-073496-4 specific region including,for example, at least 6, 7, 8, 9, 10, 15, 20, or 30 or about 7-10,10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45 nucleotides or longer.Additional primer are also set forth herein in Table 11.

As discussed elsewhere herein, any method to PCR amplify the DP-073496-4event or specific region can be employed, including for example, realtime PCR. See, for example, Livak, et al., (1995a). Oligonucleotideswith fluorescent dyes at opposite ends provide a quenched probe systemfor detecting PCR product and nucleic acid hybridization. PCR methodsand Applications. 4:357-362; U.S. Pat. No. 5,538,848; U.S. Pat. No.5,723,591; Applied Biosystems User Bulletin No. 2, “RelativeQuantitation of Gene Expression,” U.S. Pat. No. 4,303,859 and AppliedBiosystems User Bulletin No. 5, “Multiplex PCR with Taqman VIC probes,”U.S. Pat. No. 4,306,236, each of which is herein incorporated byreference.

Thus, in specific embodiments, a method of detecting the presence ofbrassica event DP-073496-4 or progeny thereof in a biological sample isprovided. The method comprises (a) extracting a DNA sample from thebiological sample; (b) providing a pair of DNA primer moleculestargeting the insert and/or junction (c) providing DNA amplificationreaction conditions; (d) performing the DNA amplification reaction,thereby producing a DNA amplicon molecule and (e) detecting the DNAamplicon molecule, wherein the detection of said DNA amplicon moleculein the DNA amplification reaction indicates the presence of Brassicaevent DP-073496-4. In order for a nucleic acid molecule to serve as aprimer or probe it needs only be sufficiently complementary in sequenceto be able to form a stable double-stranded structure under theparticular solvent and salt concentrations employed.

In hybridization techniques, all or part of a polynucleotide thatselectively hybridizes to a target polynucleotide having a DP-073496-4specific event is employed. By “stringent conditions” or “stringenthybridization conditions” when referring to a polynucleotide probeconditions under which a probe will hybridize to its target sequence toa detectably greater degree than to other sequences (e.g., at least2-fold over background) are intended. Regarding the amplification of atarget polynucleotide (e.g., by PCR) using a particular amplificationprimer pair, “stringent conditions” are conditions that permit theprimer pair to hybridize to the target polynucleotide to which oneprimer having the corresponding wild-type sequence and another primerhaving the corresponding DP-073496-4 inserted DNA sequence. Stringentconditions are sequence-dependent and will be variable in differentcircumstances. By controlling the stringency of the hybridization and/orwashing conditions, target sequences that are 100% complementary to theprobe can be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of identity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length or lessthan 500 nucleotides in length.

As used herein, a substantially identical or complementary sequence is apolynucleotide that will specifically hybridize to the complement of thenucleic acid molecule to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.Typically, stringent conditions for hybridization and detection will bethose in which the salt concentration is less than about 1.5 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. Exemplary low stringency conditions include hybridizationwith a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodiumdodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderatestringency conditions include hybridization in 40 to 45% formamide, 1.0M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C. and a wash in 0.1×SSC at 60 to 65°C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours. The duration of the wash time will be atleast a length of time sufficient to reach equilibrium.

In hybridization reactions, specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. For DNA-DNA hybrids, theT_(m) can be approximated from the equation of Meinkoth and Wahl, (1984)Anal. Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61(% form)−500/L; where M is the molarity of monovalent cations, % GC isthe percentage of guanosine and cytosine nucleotides in the DNA, % formis the percentage of formamide in the hybridization solution and L isthe length of the hybrid in base pairs. The T_(m) is the temperature(under defined ionic strength and pH) at which 50% of a complementarytarget sequence hybridizes to a perfectly matched probe. T_(m) isreduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with ≧90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9 or 10° C. lower than the thermal melting point (T_(m)); low stringencyconditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15or 20° C. lower than the thermal melting point (T_(m)). Using theequation, hybridization and wash compositions, and desired T_(m), thoseof ordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T_(m) of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is optimal toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, (1993) Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, New York) and Ausubel, et al., eds. (1995) Current Protocolsin Molecular Biology, Chapter 2 (Greene Publishing andWiley-Interscience, New York). See, Sambrook, et al., (1989) MolecularCloning: A Laboratory Manual (2d ed., Cold Spring Harbor LaboratoryPress, Plainview, N.Y.) and Haymes, et al., (1985) In: Nucleic AcidHybridization, a Practical Approach, IRL Press, Washington, D.C.

A polynucleotide is said to be the “complement” of anotherpolynucleotide if they exhibit complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the polynucleotide molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions.

Further provided are methods of detecting the presence of DNAcorresponding to the DP-073496-4 event in a sample. In one embodiment,the method comprises (a) contacting the biological sample with apolynucleotide probe that hybridizes under stringent hybridizationconditions with DNA from brassica event DP-073496-4 and specificallydetects the DP-073496-4 event; (b) subjecting the sample and probe tostringent hybridization conditions and (c) detecting hybridization ofthe probe to the DNA, wherein detection of hybridization indicates thepresence of the DP-073496-4 event.

Various methods can be used to detect the DP-073496-4 specific region oramplicon thereof, including, but not limited to, Genetic Bit Analysis(Nikiforov, et al., (1994) Nucleic Acid Res. 22:4167-4175) where a DNAoligonucleotide is designed which overlaps both the adjacent flankingDNA sequence and the inserted DNA sequence. The oligonucleotide isimmobilized in wells of a microwell plate. Following PCR of the regionof interest (using one primer in the inserted sequence and one in theadjacent flanking sequence) a single-stranded PCR product can beannealed to the immobilized oligonucleotide and serve as a template fora single base extension reaction using a DNA polymerase and labeledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization and single baseextension.

Another detection method is the Pyrosequencing technique as described byWinge, ((2000) Innov. Pharma. Tech. 00:18-24). In this method, anoligonucleotide is designed that overlaps the adjacent DNA and insertDNA junction. The oligonucleotide is annealed to a single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking sequence) and incubated in the presence of a DNApolymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. dNTPs are added individually and theincorporation results in a light signal which is measured. A lightsignal indicates the presence of the transgene insert/flanking sequencedue to successful amplification, hybridization and single or multi-baseextension.

Fluorescence Polarization as described by Chen, et al., ((1999) GenomeRes. 9:492-498) is also a method that can be used to detect an ampliconof the invention. Using this method, an oligonucleotide is designedwhich overlaps the flanking and inserted DNA junction. Theoligonucleotide is hybridized to a single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking DNA sequence) and incubated in the presence of a DNA polymeraseand a fluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the transgene insert/flanking sequence due to successfulamplification, hybridization and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps theflanking and insert DNA junction. The FRET probe and PCR primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi, et al., ((1996) Nature Biotech. 14:303-308).Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankingsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Following successful PCR amplification, hybridization of the FRETprobe to the target sequence results in the removal of the probesecondary structure and spatial separation of the fluorescent andquenching moieties. A fluorescent signal results. A fluorescent signalindicates the presence of the flanking/transgene insert sequence due tosuccessful amplification and hybridization.

A hybridization reaction using a probe specific to a sequence foundwithin the amplicon is yet another method used to detect the ampliconproduced by a PCR reaction.

As used herein, “kit” refers to a set of reagents for the purpose ofperforming the method embodiments of the invention, more particularly,the identification and/or the detection of the DP-073496-4 event inbiological samples. The kit of the invention can be used and itscomponents can be specifically adjusted, for purposes of quality control(e.g. purity of seed lots), detection of event DP-073496-4 in plantmaterial or material comprising or derived from plant material, such asbut not limited to food or feed products.

In specific embodiments, a kit for identifying event DP-073496-4 in abiological sample is provided. The kit comprises a first and a secondprimer, wherein the first and second primer amplify a polynucleotidecomprising a DP-073496-4 specific region. In further embodiments, thekit also comprises a polynucleotide for the detection of the DP-073496-4specific region. The kit can comprise, for example, a first primercomprising a fragment of a polynucleotide of SEQ ID NO: 2, 3, 8, 9, or10, wherein the first or the second primer shares sufficient sequencehomology or complementarity and specificity to the polynucleotide toamplify said DP-073496-4 specific region. For example, in specificembodiments, the first primer comprises a fragment of a polynucleotideof SEQ ID NO: 2 or 3, wherein the first or the second primer sharessufficient sequence homology or complementarity to the polynucleotide toamplify the DP-073496-4 specific region. In other embodiments, the firstprimer comprises a fragment of a polynucleotide of SEQ ID NO: 8 and thesecond primer comprises a fragment of SEQ ID NO: 9 or 10, wherein thefirst or the second primer shares sufficient sequence homology orcomplementarity to the polynucleotide to amplify the DP061061-7 specificregion. Alternatively, the first primer pair comprises SEQ ID NO:9 or avariant or fragment thereof and the second primer comprises SEQ ID NO: 8or 10 or a variant or fragment thereof. In other embodiments, the primerpair can comprise a fragment of SEQ ID NO: 2 and a fragment of SEQ IDNO: 3. The primers can be of any length sufficient to amplify theDP-073496-4 region including, for example, at least 6, 7, 8, 9, 10, 15,20, 15 or 30 or about 7-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40,40-45 nucleotides or longer. Further provided are DNA detection kitscomprising at least one polynucleotide that can specifically detect aDP-073496-4 specific region or insert DNA, wherein said polynucleotidecomprises at least one DNA molecule of a sufficient length of contiguousnucleotides homologous or complementary to SEQ ID NO: 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 19, 20, 21, 22, 23, 24, 25, 26,or 27.

In one embodiment, a kit for identifying event DP-073496-4 in abiological sample is provided. The kit comprises a first and a secondprimer, wherein said first and said second primer amplify apolynucleotide comprising a DP-073496-4 specific region. In furtherembodiments, the kit further comprises a polynucleotide for thedetection of the DP-073496-4 specific region. Thus, in one non-limitingembodiment, the first primer comprises a first fragment of SEQ ID NO: 11and the second primer comprises a second fragment of SEQ ID NO:11,wherein the first and the second primer flank the DP-073496-4 specificregion and share sufficient sequence homology or complementarity to thepolynucleotide to amplify said DP-073496-4 specific region. As such, akit can therefore include a first primer comprising a fragment of SEQ IDNO:8 and a second primer comprising a fragment of SEQ ID NO:9; or afirst or a second primer comprising at least 8 consecutivepolynucleotides of SEQ ID NO: 11; or a first or a second primercomprising at least 8 consecutive polynucleotides of SEQ ID NO:8 or 9.

In further embodiments, methods are provided for detecting aglyphosate-N-acetyltranferase polypeptide comprising analysing brassicaplant tissues using an immunoassay comprising aglyphosate-N-acetyltranferase polypeptide-specific antibody orantibodies. In other embodiments, methods for detecting the presence ofa polynucleotide that encodes a glyphosate-N-acetyltranferasepolypeptide are provide and comprise assaying brassica plant tissueusing PCR amplification. Kits for employing such methods are furtherprovided.

Any of the polynucleotides and fragments and variants thereof employedin the methods and compositions of the invention can share sequenceidentity to a region of the transgene insert of the DP-073496-4 event, ajunction sequence of the DP-073496-4 event, or a region of the insert incombination with a region of the flanking sequence of the DP-073496-4event. Methods to determine the relationship of various sequences areknown. As used herein, “reference sequence” is a defined sequence usedas a basis for sequence comparison. A reference sequence may be a subsetor the entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence. As used herein, “comparison window” makes reference to acontiguous and specified segment of a polynucleotide sequence, whereinthe polynucleotide sequence in the comparison window may compriseadditions or deletions (i.e., gaps) compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the two polynucleotides. Generally, the comparison window is at least20 contiguous nucleotides in length and optionally can be 30, 40, 50,100 or longer. Those of skill in the art understand that to avoid a highsimilarity to a reference sequence due to inclusion of gaps in thepolynucleotide sequence a gap penalty is typically introduced and issubtracted from the number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, (1988) CABIOS 4:11-17; the local alignmentalgorithm of Smith, et al., (1981) Adv. Appl. Math. 2:482; the globalalignment algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-453; the search-for-local alignment method of Pearson and Lipman,(1988) Proc. Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin andAltschul, (1990) Proc. Natl. Acad. Sci. USA 872264, modified as inKarlin and Altschul, (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins, et al.,(1988) Gene 73:237-244 (1988); Higgins, et al., (1989) CABIOS 5:151-153;Corpet, et al., (1988) Nucleic Acids Res. 16:10881-90; Huang, et al.,(1992) CABIOS 8:155-65 and Pearson, et al., (1994) Meth. Mol. Biol.24:307-331. The ALIGN program is based on the algorithm of Myers andMiller, (1988) supra. A PAM120 weight residue table, a gap lengthpenalty of 12 and a gap penalty of 4 can be used with the ALIGN programwhen comparing amino acid sequences. The BLAST programs of Altschul, etal., (1990) J. Mol. Biol. 215:403 are based on the algorithm of Karlinand Altschul, (1990) supra. BLAST nucleotide searches can be performedwith the BLASTN program, score=100, wordlength=12, to obtain nucleotidesequences homologous to a nucleotide sequence encoding a protein of theinvention. BLAST protein searches can be performed with the BLASTXprogram, score=50, wordlength=3, to obtain amino acid sequenceshomologous to a protein or polypeptide of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0)can be utilized as described in Altschul, et al., (1997) Nucleic AcidsRes. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used toperform an iterated search that detects distant relationships betweenmolecules. See, Altschul, et al., (1997) supra. When utilizing BLAST,Gapped BLAST, PSI-BLAST, the default parameters of the respectiveprograms (e.g., BLASTN for nucleotide sequences, BLASTX for proteins)can be used. See www.ncbi.nlm.nih.gov. Alignment may also be performedmanually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2 and theBLOSUM62 scoring matrix or any equivalent program thereof. By“equivalent program” any sequence comparison program that, for any twosequences in question, generates an alignment having identicalnucleotide or amino acid residue matches and an identical percentsequence identity when compared to the corresponding alignment generatedby GAP Version 10 is intended.

GAP uses the algorithm of Needleman and Wunsch, (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity and Similarity. The Quality is the metric maximized in order toalign the sequences. Ratio is the Quality divided by the number of basesin the shorter segment. Percent Identity is the percent of the symbolsthat actually match. Percent Similarity is the percent of the symbolsthat are similar. Symbols that are across from gaps are ignored. Asimilarity is scored when the scoring matrix value for a pair of symbolsis greater than or equal to 0.50, the similarity threshold. The scoringmatrix used in Version 10 of the GCG Wisconsin Genetics Software Packageis BLOSUM62 (see, Henikoff and Henikoff, (1989) Proc. Natl. Acad. Sci.USA 89:10915).

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison and multiplying the result by 100 to yield the percentage ofsequence identity.

The present invention provides methods for controlling weeds in an areaof cultivation, preventing the development or the appearance ofherbicide resistant weeds in an area of cultivation, producing a cropand increasing crop safety. The term “controlling,” and derivationsthereof, for example, as in “controlling weeds” refers to one or more ofinhibiting the growth, germination, reproduction and/or proliferation ofand/or killing, removing, destroying or otherwise diminishing theoccurrence and/or activity of a weed.

As used herein, an “area of cultivation” comprises any region in whichone desires to grow a plant. Such areas of cultivations include, but arenot limited to, a field in which a plant is cultivated (such as a cropfield, a sod field, a tree field, a managed forest, a field forculturing fruits and vegetables, etc), a greenhouse, a growth chamber,etc.

The methods of the invention comprise planting the area of cultivationwith the Brassica DP-073496-4 seeds or plants, and in specificembodiments, applying to the crop, seed, weed or area of cultivationthereof an effective amount of a herbicide of interest. It is recognizedthat the herbicide can be applied before or after the crop is planted inthe area of cultivation. Such herbicide applications can include anapplication of glyphosate.

In one embodiment, the method of controlling weeds comprises plantingthe area with the DP-073496-4 Brassica seeds or plants and applying tothe crop, crop part, seed of said crop or the area under cultivation, aneffective amount of a herbicide, wherein said effective amount comprisesan amount that is not tolerated by a second control crop when applied tothe second crop, crop part, seed or the area of cultivation, whereinsaid second control crop does not express the GLYAT polynucleotide.

In another embodiment, the method of controlling weeds comprisesplanting the area with a DP-073496-4 Brassica crop seed or plant andapplying to the crop, crop part, seed of said crop or the area undercultivation, an effective amount of a glyphosate herbicide, wherein saideffective amount comprises a level that is above the recommended labeluse rate for the crop, wherein said effective amount is tolerated whenapplied to the DP-073496-4 Brassica crop, crop part, seed or the area ofcultivation thereof.

A “control” or “control plant” or “control plant cell” provides areference point for measuring changes in phenotype of the subject plantor plant cell, and may be any suitable plant or plant cell. A controlplant or plant cell may comprise, for example: (a) a wild-type plant orcell, i.e., of the same genotype as the starting material for thegenetic alteration which resulted in the subject plant or cell; (b) aplant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell which is genetically identical to thesubject plant or plant cell but which is not exposed to the sametreatment (e.g., herbicide treatment) as the subject plant or plantcell; (e) the subject plant or plant cell itself, under conditions inwhich the gene of interest is not expressed or (f) the subject plant orplant cell itself, under conditions in which it has not been exposed toa particular treatment such as, for example, a herbicide or combinationof herbicides and/or other chemicals. In some instances, an appropriatecontrol plant or control plant cell may have a different genotype fromthe subject plant or plant cell but may share the herbicide-sensitivecharacteristics of the starting material for the genetic alteration(s)which resulted in the subject plant or cell (see, e.g., Green, (1998)Weed Technology 12:474-477; Green and Ulrich, (1993) Weed Science41:508-516. In other embodiments, the null segregant can be used as acontrol, as they are genetically identical to DP-073496-4 with theexception of the transgenic insert DNA.

Classification of herbicides (i.e., the grouping of herbicides intoclasses and subclasses) is well-known in the art and includesclassifications by HRAC (Herbicide Resistance Action Committee) and WSSA(the Weed Science Society of America) (see also, Retzinger andMallory-Smith, (1997) Weed Technology 11:384-393). An abbreviatedversion of the HRAC classification (with notes regarding thecorresponding WSSA group) is set forth below in Table 1.

Herbicides can be classified by their mode of action and/or site ofaction and can also be classified by the time at which they are applied(e.g., preemergent or postemergent), by the method of application (e.g.,foliar application or soil application) or by how they are taken up byor affect the plant. For example, thifensulfuron-methyl andtribenuron-methyl are applied to the foliage of a crop and are generallymetabolized there, while rimsulfuron and chlorimuron-ethyl are generallytaken up through both the roots and foliage of a plant. “Mode of action”generally refers to the metabolic or physiological process within theplant that the herbicide inhibits or otherwise impairs, whereas “site ofaction” generally refers to the physical location or biochemical sitewithin the plant where the herbicide acts or directly interacts.Herbicides can be classified in various ways, including by mode ofaction and/or site of action (see, e.g., Table 1).

Often, a herbicide-tolerance gene that confers tolerance to a particularherbicide or other chemical on a plant expressing it will also confertolerance to other herbicides or chemicals in the same class orsubclass, for example, a class or subclass set forth in Table 1. Thus,in some embodiments of the invention, a transgenic plant of theinvention is tolerant to more than one herbicide or chemical in the sameclass or subclass, such as, for example, an inhibitor of PPO, asulfonylurea or a synthetic auxin.

Typically, the plants of the present invention can tolerate treatmentwith different types of herbicides (i.e., herbicides having differentmodes of action and/or different sites of action) as well as with higheramounts of herbicides than previously known plants, thereby permittingimproved weed management strategies that are recommended in order toreduce the incidence and prevalence of herbicide-tolerant weeds.Specific herbicide combinations can be employed to effectively controlweeds.

The invention thereby provides a transgenic brassica plant which can beselected for use in crop production based on the prevalence ofherbicide-tolerant weed species in the area where the transgenic crop isto be grown. Methods are known in the art for assessing the herbicidetolerance of various weed species. Weed management techniques are alsoknown in the art, such as for example, crop rotation using a crop thatis tolerant to a herbicide to which the local weed species are nottolerant. A number of entities monitor and publicly report the incidenceand characteristics of herbicide-tolerant weeds, including the HerbicideResistance Action Committee (HRAC), the Weed Science Society of Americaand various state agencies (see, for example, herbicide tolerance scoresfor various broadleaf weeds from the 2004 Illinois Agricultural PestManagement Handbook) and one of skill in the art would be able to usethis information to determine which crop and herbicide combinationsshould be used in a particular location.

These entities also publish advice and guidelines for preventing thedevelopment and/or appearance of and controlling the spread of herbicidetolerant weeds (see, e.g., Owen and Hartzler, (2004), 2005 HerbicideManual for Agricultural Professionals, Pub. WC 92 Revised (Iowa StateUniversity Extension, Iowa State University of Science and Technology,Ames, Iowa); Weed Control for Corn, Brassicas, and Sorghum, Chapter 2 of“2004 Illinois Agricultural Pest Management Handbook” (University ofIllinois Extension, University of Illinois at Urbana-Champaign, Ill.);Weed Control Guide for Field Crops, MSU Extension Bulletin E434(Michigan State University, East Lansing, Mich.)).

TABLE 1 Abbreviated version of HRAC Herbicide Classification I. ALSInhibitors (WSSA Group 2) A. Sulfonylureas 1. Azimsulfuron 2.Chlorimuron-ethyl 3. Metsulfuron-methyl 4. Nicosulfuron 5. Rimsulfuron6. Sulfometuron-methyl 7. Thifensulfuron-methyl 8. Tribenuron-methyl 9.Amidosulfuron 10. Bensulfuron-methyl 11. Chlorsulfuron 12. Cinosulfuron13. Cyclosulfamuron 14. Ethametsulfuron-methyl 15. Ethoxysulfuron 16.Flazasulfuron 17. Flupyrsulfuron-methyl 18. Foramsulfuron 19.Imazosulfuron 20. Iodosulfuron-methyl 21. Mesosulfuron-methyl 22.Oxasulfuron 23. Primisulfuron-methyl 24. Prosulfuron 25.Pyrazosulfuron-ethyl 26. Sulfosulfuron 27. Triasulfuron 28.Trifloxysulfuron 29. Triflusulfuron-methyl 30. Tritosulfuron 31.Halosulfuron-methyl 32. Flucetosulfuron B.Sulfonylaminocarbonyltriazolinones 1. Flucarbazone 2. Procarbazone C.Triazolopyrimidines 1. Cloransulam-methyl 2. Flumetsulam 3. Diclosulam4. Florasulam 5. Metosulam 6. Penoxsulam 7. Pyroxsulam D.Pyrimidinyloxy(thio)benzoates 1. Bispyribac 2. Pyriftalid 3.Pyribenzoxim 4. Pyrithiobac 5. Pyriminobac-methyl E. Imidazolinones 1.Imazapyr 2. Imazethapyr 3. Imazaquin 4. Imazapic 5.Imazamethabenz-methyl 6. Imazamox II. Other Herbicides--ActiveIngredients/Additional Modes of Action A. Inhibitors of Acetyl CoAcarboxylase (ACCase) (WSSA Group 1) 1. Aryloxyphenoxypropionates(‘FOPs’) a. Quizalofop-P-ethyl b. Diclofop-methyl c.Clodinafop-propargyl d. Fenoxaprop-P-ethyl e. Fluazifop-P-butyl f.Propaquizafop g. Haloxyfop-P-methyl h. Cyhalofop-butyl i.Quizalofop-P-ethyl 2. Cyclohexanediones (‘DIMs’) a. Alloxydim b.Butroxydim c. Clethodim d. Cycloxydim e. Sethoxydim f. Tepraloxydim g.Tralkoxydim B. Inhibitors of Photosystem II-HRAC Group C1/WSSA Group5 1. Triazines a. Ametryne b. Atrazine c. Cyanazine d. Desmetryne e.Dimethametryne f. Prometon g. Prometryne h. Propazine i. Simazine j.Simetryne k. Terbumeton l. Terbuthylazine m. Terbutryne n. Trietazine 2.Triazinones a. Hexazinone b. Metribuzin c. Metamitron 3. Triazolinone a.Amicarbazone 4. Uracils a. Bromacil b. Lenacil c. Terbacil 5.Pyridazinones a. Pyrazon 6. Phenyl carbamates a. Desmedipham b.Phenmedipham C. Inhibitors of Photosystem II--HRAC Group C2/WSSA Group7 1. Ureas a. Fluometuron b. Linuron c. Chlorobromuron d. Chlorotolurone. Chloroxuron f. Dimefuron g. Diuron h. Ethidimuron i. Fenuron j.Isoproturon k. Isouron l. Methabenzthiazuron m. Metobromuron n.Metoxuron o. Monolinuron p. Neburon q. Siduron r. Tebuthiuron 2. Amidesa. Propanil b. Pentanochlor D. Inhibitors of Photosystem II--HRAC GroupC3/WSSA Group 6 1. Nitriles a. Bromofenoxim b. Bromoxynil c. Ioxynil 2.Benzothiadiazinone (Bentazon) a. Bentazon 3. Phenylpyridazines a.Pyridate b. Pyridafol E. Photosystem-l-electron diversion(Bipyridyliums) (WSSA Group 22) 1. Diquat 2. Paraquat F. Inhibitors ofPPO (protoporphyrinogen oxidase) (WSSA Group 14) 1. Diphenylethers a.Acifluorfen-Na b. Bifenox c. Chlomethoxyfen d. Fluoroglycofen-ethyl e.Fomesafen f. Halosafen g. Lactofen h. Oxyfluorfen 2. Phenylpyrazoles a.Fluazolate b. Pyraflufen-ethyl 3. N-phenylphthalimides a. Cinidon-ethylb. Flumioxazin c. Flumiclorac-pentyl 4. Thiadiazoles a.Fluthiacet-methyl b. Thidiazimin 5. Oxadiazoles a. Oxadiazon b.Oxadiargyl 6. Triazolinones a. Carfentrazone-ethyl b. Sulfentrazone 7.Oxazolidinediones a. Pentoxazone 8. Pyrimidindiones a. Benzfendizone b.Butafenicil 9. Others a. Pyrazogyl b. Profluazol G. Bleaching:Inhibition of carotenoid biosynthesis at the phytoene desaturase step(PDS) (WSSA Group 12) 1. Pyridazinones a. Norflurazon 2.Pyridinecarboxamides a. Diflufenican b. Picolinafen 3. Others a.Beflubutamid b. Fluridone c. Flurochloridone d. Flurtamone H. Bleaching:Inhibition of 4-hydroxyphenyl-pyruvate-dioxygenase (4-HPPD) (WSSA Group28) 1. Triketones a. Mesotrione b. Sulcotrione c. topremezone d.temtorione 2. Isoxazoles a. Isoxachlortole b. Isoxaflutole 3. Pyrazolesa. Benzofenap b. Pyrazoxyfen c. Pyrazolynate 4. Others a. BenzobicyclonI. Bleaching: Inhibition of carotenoid biosynthesis (unknown target)(WSSA Group 11 and 13) 1. Triazoles (WSSA Group 11) a. Amitrole 2.Isoxazolidinones (WSSA Group 13) a. Clomazone 3. Ureas a. Fluometuron 3.Diphenylether a. Aclonifen J. Inhibition of EPSP Synthase 1. Glycines(WSSA Group 9) a. Glyphosate b. Sulfosate K. Inhibition of glutaminesynthetase 1. Phosphinic Acids a. Glufosinate-ammonium b. Bialaphos L.Inhibition of DHP (dihydropteroate) synthase (WSSA Group 18) 1Carbamates a. Asulam M. Microtubule Assembly Inhibition (WSSA Group3) 1. Dinitroanilines a. Benfluralin b. Butralin c. Dinitramine d.Ethalfluralin e. Oryzalin f. Pendimethalin g. Trifluralin 2.Phosphoroamidates a. Amiprophos-methyl b. Butamiphos 3. Pyridines a.Dithiopyr b. Thiazopyr 4. Benzamides a. Pronamide b. Tebutam 5.Benzenedicarboxylic acids a. Chlorthal-dimethyl N. Inhibition ofmitosis/microtubule organization WSSA Group 23) 1. Carbamates a.Chlorpropham b. Propham c. Carbetamide O. Inhibition of cell division(Inhibition of very long chain fatty acids as proposed mechanism; WSSAGroup 15) 1. Chloroacetamides a. Acetochlor b. Alachlor c. Butachlor d.Dimethachlor e. Dimethanamid f. Metazachlor g. Metolachlor h. Pethoxamidi. Pretilachlor j. Propachlor k. Propisochlor l. Thenylchlor 2.Acetamides a. Diphenamid b. Napropamide c. Naproanilide 3. Oxyacetamidesa. Flufenacet b. Mefenacet 4. Tetrazolinones a. Fentrazamide 5. Othersa. Anilofos b. Cafenstrole c. Indanofan d. Piperophos P. Inhibition ofcell wall (cellulose) synthesis 1. Nitriles (WSSA Group 20) a.Dichlobenil b. Chlorthiamid 2. Benzamides (isoxaben (WSSA Group 21)) a.Isoxaben 3. Triazolocarboxamides (flupoxam) a. Flupoxam Q. Uncoupling(membrane disruption): (WSSA Group 24) 1. Dinitrophenols a. DNOC b.Dinoseb c. Dinoterb R. Inhibition of Lipid Synthesis by other than ACCinhibition 1. Thiocarbamates (WSSA Group 8) a. Butylate b. Cycloate c.Dimepiperate d. EPTC e. Esprocarb f. Molinate g. Orbencarb h. Pebulatei. Prosulfocarb j. Benthiocarb k. Tiocarbazil l. Triallate m. Vernolate2. Phosphorodithioates a. Bensulide 3. Benzofurans a. Benfuresate b.Ethofumesate 4. Halogenated alkanoic acids (WSSA Group 26) a. TCA b.Dalapon c. Flupropanate S. Synthetic auxins (IAA-like) (WSSA Group 4) 1.Phenoxycarboxylic acids a. Clomeprop b. 2,4-D c. Mecoprop 2. Benzoicacids a. Dicamba b. Chloramben c. TBA 3. Pyridine carboxylic acids a.Clopyralid b. Fluroxypyr c. Picloram d. Tricyclopyr 4. Quinolinecarboxylic acids a. Quinclorac b. Quinmerac 5. Others (benazolin-ethyl)a. Benazolin-ethyl T. Inhibition of Auxin Transport 1. Phthalamates;semicarbazones (WSSA Group 19) a. Naptalam b. Diflufenzopyr-Na U. OtherMechanism of Action 1. Arylaminopropionic acids a.Flamprop-M-methyl/-isopropyl 2. Pyrazolium a. Difenzoquat 3.Organoarsenicals a. DSMA b. MSMA 4. Others a. Bromobutide b. Cinmethylinc. Cumyluron d. Dazomet e. Daimuron-methyl f. Dimuron g. Etobenzanid h.Fosamine i. Metam j. Oxaziclomefone k. Oleic acid l. Pelargonic acid m.Pyributicarb

In certain methods, glyphosate, alone or in combination with anotherherbicide of interest, can be applied to the DP-073496-4 Brassica plantsor their area of cultivation. Non-limiting examples of glyphosateformations are set forth in Table 2. In specific embodiments, theglyphosate is in the form of a salt, such as, ammonium,isopropylammonium, potassium, sodium (including sesquisodium) ortrimesium (alternatively named sulfosate).

TABLE 2 Glyphosate formulations comparisons. Active Acid Apply: AcidHerbicide by ingredient equivalent fl oz/ equivalent RegisteredTrademark Manufacturer Salt per gallon per gallon acre per acre RoundupOriginal Monsanto Isopropylamine 4 3 32 0.750 Roundup Original IIMonsanto Isopropylamine 4 3 32 0.750 Roundup Original MAX MonsantoPotassium 5.5 4.5 22 0.773 Roundup UltraMax Monsanto Isopropylamine 53.68 26 0.748 Roundup UltraMax II Monsanto Potassium 5.5 4.5 22 0.773Roundup Weathermax Monsanto Potassium 5.5 4.5 22 0.773 TouchdownSyngenta Diammonium 3.7 3 32 0.750 Touchdown HiTech Syngenta Potassium6.16 5 20 0.781 Touchdown Total Syngenta Potassium 5.14 4.17 24 0.782Durango Dow AgroSciences Isopropylamine 5.4 4 24 0.750 Glyphomax DowAgroSciences Isopropylamine 4 3 32 0.750 Glyphomax Plus Dow AgroSciencesIsopropylamine 4 3 32 0.750 Glyphomax XRT Dow AgroSciencesIsopropylamine 4 3 32 0.750 Gly Star Plus Albaugh/Agri StarIsopropylamine 4 3 32 0.750 Gly Star 5 Albaugh/Agri Star Isopropylamine5.4 4 24 0.750 Gly Star Original Albaugh/Agri Star Isopropylamine 4 3 320.750 Gly-Flo Micro Flo Isopropylamine 4 3 32 0.750 Credit NufarmIsopropylamine 4 3 32 0.750 Credit Extra Nufarm Isopropylamine 4 3 320.750 Credit Duo Nufarm Isopro. + monoamm. 4 3 32 0.750 Credit Duo ExtraNufarm Isopro. + monoamm. 4 3 32 0.750 Extra Credit 5 NufarmIsopropylamine 5 3.68 26 0.748 Cornerstone Agriliance Isopropylamine 4 332 0.750 Cornerstone Plus Agriliance Isopropylamine 4 3 32 0.750 GlyfosCheminova Isopropylamine 4 3 32 0.750 Glyfos X-TRA CheminovaIsopropylamine 4 3 32 0.750 Rattler Helena Isopropylamine 4 3 32 0.750Rattler Plus Helena Isopropylamine 4 3 32 0.750 Mirage UAPIsopropylamine 4 3 32 0.750 Mirage Plus UAP Isopropylamine 4 3 32 0.750Glyphosate 41% Helm Agro USA Isopropylamine 4 3 32 0.750 BuccaneerTenkoz Isopropylamine 4 3 32 0.750 Buccaneer Plus Tenkoz Isopropylamine4 3 32 0.750 Honcho Monsanto Isopropylamine 4 3 32 0.750 Honcho PlusMonsanto Isopropylamine 4 3 32 0.750 Gly-4 Univ. Crop Prot. Alli.Isopropylamine 4 3 32 0.750 Gly-4 Plus Univ. Crop Prot. Alli.Isopropylamine 4 3 32 0.750 ClearOut 41 Chemical Products Tech.Isopropylamine 4 3 32 0.750 ClearOut 41 Plus Chemical Products Tech.Isopropylamine 4 3 32 0.750 Spitfire Control Soultions Isopropylamine 43 32 0.750 Spitfire Plus Control Soultions Isopropylamine 4 3 32 0.750Glyphosate 4 FarmerSaver.com Isopropylamine 4 3 32 0.750 FS GlyphosatePlus Growmark Isopropylamine 4 3 32 0.750 Glyphosate Original Griffin,LLC Isopropylamine 4 3 32 0.750

Thus, in some embodiments, a transgenic plant of the invention is usedin a method of growing a DP-073496-4 brassica crop by the application ofherbicides to which the plant is tolerant. In this manner, treatmentwith a combination of one of more herbicides which include, but are notlimited to: acetochlor, acifluorfen and its sodium salt, aclonifen,acrolein (2-propenal), alachlor, alloxydim, ametryn, amicarbazone,amidosulfuron, aminopyralid, amitrole, ammonium sulfamate, anilofos,asulam, atrazine, azimsulfuron, beflubutamid, benazolin,benazolin-ethyl, bencarbazone, benfluralin, benfuresate,bensulfuron-methyl, bensulide, bentazone, benzobicyclon, benzofenap,bifenox, bilanafos, bispyribac and its sodium salt, bromacil,bromobutide, bromofenoxim, bromoxynil, bromoxynil octanoate, butachlor,butafenacil, butamifos, butralin, butroxydim, butylate, cafenstrole,carbetamide, carfentrazone-ethyl, catechin, chlomethoxyfen, chloramben,chlorbromuron, chlorflurenol-methyl, chloridazon, chlorimuron-ethyl,chlorotoluron, chlorpropham, chlorsulfuron, chlorthal-dimethyl,chlorthiamid, cinidon-ethyl, cinmethylin, cinosulfuron, clethodim,clodinafop-propargyl, clomazone, clomeprop, clopyralid,clopyralid-olamine, cloransulam-methyl, CUH-35 (2-methoxyethyl2-[[[4-chloro-2-fluoro-5-[(1-methyl-2-propynyl)oxy]phenyl](3-fluorobenzoyl)amino]carbonyl]-1-cyclohexene-1-carboxylate),cumyluron, cyanazine, cycloate, cyclosulfamuron, cycloxydim,cyhalofop-butyl, 2,4-D and its butotyl, butyl, isoctyl and isopropylesters and its dimethylammonium, diolamine and trolamine salts,daimuron, dalapon, dalapon-sodium, dazomet, 2,4-DB and itsdimethylammonium, potassium and sodium salts, desmedipham, desmetryn,dicamba and its diglycolammonium, dimethylammonium, potassium and sodiumsalts, dichlobenil, dichlorprop, diclofop-methyl, diclosulam,difenzoquat metilsulfate, diflufenican, diflufenzopyr, dimefuron,dimepiperate, dimethachlor, dimethametryn, dimethenamid, dimethenamid-P,dimethipin, dimethylarsinic acid and its sodium salt, dinitramine,dinoterb, diphenamid, diquat dibromide, dithiopyr, diuron, DNOC,endothal, EPTC, esprocarb, ethalfluralin, ethametsulfuron-methyl,ethofumesate, ethoxyfen, ethoxysulfuron, etobenzanid, fenoxaprop-ethyl,fenoxaprop-P-ethyl, fentrazamide, fenuron, fenuron-TCA, flamprop-methyl,flamprop-M-isopropyl, flamprop-M-methyl, flazasulfuron, florasulam,fluazifop-butyl, fluazifop-P-butyl, flucarbazone, flucetosulfuron,fluchloralin, flufenacet, flufenpyr, flufenpyr-ethyl, flumetsulam,flumiclorac-pentyl, flumioxazin, fluometuron, fluoroglycofen-ethyl,flupyrsulfuron-methyl and its sodium salt, flurenol, flurenol-butyl,fluridone, fluorochloridone, fluoroxypyr, flurtamone, fluthiacet-methyl,fomesafen, foramsulfuron, fosamine-ammonium, glufosinate,glufosinate-ammonium, glyphosate and its salts such as ammonium,isopropylammonium, potassium, sodium (including sesquisodium) andtrimesium (alternatively named sulfosate), halosulfuron-methyl,haloxyfop-etotyl, haloxyfop-methyl, hexazinone, HOK-201(N-(2,4-difluorophenyl)-1,5-dihydro-N-(1-methylethyl)-5-oxo-1-[(tetrahydro-2H-pyran-2-yl)methyl]-4H-1,2,4-triazole-4-carboxamide),imazamethabenz-methyl, imazamox, imazapic, imazapyr, imazaquin,imazaquin-ammonium, imazethapyr, imazethapyr-ammonium, imazosulfuron,indanofan, iodosulfuron-methyl, ioxynil, ioxynil octanoate,ioxynil-sodium, isoproturon, isouron, isoxaben, isoxaflutole,isoxachlortole, lactofen, lenacil, linuron, maleic hydrazide, MCPA andits salts (e.g., MCPA-dimethylammonium, MCPA-potassium and MCPA-sodium,esters (e.g., MCPA-2-ethylhexyl, MCPA-butotyl) and thioesters (e.g.,MCPA-thioethyl), MCPB and its salts (e.g., MCPB-sodium) and esters(e.g., MCPB-ethyl), mecoprop, mecoprop-P, mefenacet, mefluidide,mesosulfuron-methyl, mesotrione, metam-sodium, metamifop, metamitron,metazachlor, methabenzthiazuron, methylarsonic acid and its calcium,monoammonium, monosodium and disodium salts, methyldymron, metobenzuron,metobromuron, metolachlor, S-metholachlor, metosulam, metoxuron,metribuzin, metsulfuron-methyl, molinate, monolinuron, naproanilide,napropamide, naptalam, neburon, nicosulfuron, norflurazon, orbencarb,oryzalin, oxadiargyl, oxadiazon, oxasulfuron, oxaziclomefone,oxyfluorfen, paraquat dichloride, pebulate, pelargonic acid,pendimethalin, penoxsulam, pentanochlor, pentoxazone, perfluidone,pethoxyamid, phenmedipham, picloram, picloram-potassium, picolinafen,pinoxaden, piperofos, pretilachlor, primisulfuron-methyl, prodiamine,profoxydim, prometon, prometryn, propachlor, propanil, propaquizafop,propazine, propham, propisochlor, propoxycarbazone, propyzamide,prosulfocarb, prosulfuron, pyraclonil, pyraflufen-ethyl, pyrasulfotole,pyrazogyl, pyrazolynate, pyrazoxyfen, pyrazosulfuron-ethyl,pyribenzoxim, pyributicarb, pyridate, pyriftalid, pyriminobac-methyl,pyrimisulfan, pyrithiobac, pyrithiobac-sodium, pyroxsulam, quinclorac,quinmerac, quinoclamine, quizalofop-ethyl, quizalofop-P-ethyl,quizalofop-P-tefuryl, rimsulfuron, sethoxydim, siduron, simazine,simetryn, sulcotrione, sulfentrazone, sulfometuron-methyl,sulfosulfuron, 2,3,6-TBA, TCA, TCA-sodium, tebutam, tebuthiuron,tefuryltrione, tembotrione, tepraloxydim, terbacil, terbumeton,terbuthylazine, terbutryn, thenylchlor, thiazopyr, thiencarbazone,thifensulfuron-methyl, thiobencarb, tiocarbazil, topramezone,tralkoxydim, tri-allate, triasulfuron, triaziflam, tribenuron-methyl,triclopyr, triclopyr-butotyl, triclopyr-triethylammonium, tridiphane,trietazine, trifloxysulfuron, trifluralin, triflusulfuron-methyl,tritosulfuron and vernolate is disclosed.

Other suitable herbicides and agricultural chemicals are known in theart, such as, for example, those described in WO 2005/041654. Otherherbicides also include bioherbicides such as Alternaria destruensSimmons, Colletotrichum gloeosporiodes (Penz.) Penz. and Sacc.,Drechsiera monoceras (MTB-951), Myrothecium verrucaria (Albertini &Schweinitz) Ditmar: Fries, Phytophthora palmivora (Butl.) Butyl. andPuccinia thlaspeos Schub. Combinations of various herbicides can resultin a greater-than-additive (i.e., synergistic) effect on weeds and/or aless-than-additive effect (i.e., safening) on crops or other desirableplants. In certain instances, combinations of glyphosate with otherherbicides having a similar spectrum of control but a different mode ofaction will be particularly advantageous for preventing the developmentof resistant weeds. Herbicidally effective amounts of any particularherbicide can be easily determined by one skilled in the art throughsimple experimentation.

Herbicides may be classified into groups and/or subgroups as describedherein above with reference to their mode of action, or they may beclassified into groups and/or subgroups in accordance with theirchemical structure.

Sulfonamide herbicides have as an essential molecular structure featurea sulfonamide moiety (—S(O)₂NH—). As referred to herein, sulfonamideherbicides particularly comprise sulfonylurea herbicides,sulfonylaminocarbonyltriazolinone herbicides and triazolopyrimidineherbicides. In sulfonylurea herbicides the sulfonamide moiety is acomponent in a sulfonylurea bridge (—S(O)₂NHC(O)NH(R)—). In sulfonylureaherbicides the sulfonyl end of the sulfonylurea bridge is connectedeither directly or by way of an oxygen atom or an optionally substitutedamino or methylene group to a typically substituted cyclic or acyclicgroup. At the opposite end of the sulfonylurea bridge, the amino group,which may have a substituent such as methyl (R being CH₃) instead ofhydrogen, is connected to a heterocyclic group, typically a symmetricpyrimidine or triazine ring, having one or two substituents such asmethyl, ethyl, trifluoromethyl, methoxy, ethoxy, methylamino,dimethylamino, ethylamino and the halogens. Insulfonylaminocarbonyltriazolinone herbicides, the sulfonamide moiety isa component of a sulfonylaminocarbonyl bridge (—S(O)₂NHC(O)—). Insulfonylaminocarbonyltriazolinone herbicides the sulfonyl end of thesulfonylaminocarbonyl bridge is typically connected to substitutedphenyl ring. At the opposite end of the sulfonylaminocarbonyl bridge,the carbonyl is connected to the 1-position of a triazolinone ring,which is typically substituted with groups such as alkyl and alkoxy. Intriazolopyrimidine herbicides the sulfonyl end of the sulfonamide moietyis connected to the 2-position of a substituted[1,2,4]triazolopyrimidine ring system and the amino end of thesulfonamide moiety is connected to a substituted aryl, typically phenyl,group or alternatively the amino end of the sulfonamide moiety isconnected to the 2-position of a substituted [1,2,4]triazolopyrimidinering system and the sulfonyl end of the sulfonamide moiety is connectedto a substituted aryl, typically pyridinyl, group.

The methods further comprise applying to the crop and the weeds in afield a sufficient amount of at least one herbicide to which the cropseeds or plants are tolerant, such as, for example, glyphosate, ahydroxyphenylpyruvatedioxygenase inhibitor (e.g., mesotrione orsulcotrione), a phytoene desaturase inhibitor (e.g., diflufenican), apigment synthesis inhibitor, sulfonamide, imidazolinone, bialaphos,phosphinothricin, azafenidin, butafenacil, sulfosate, glufosinate,triazolopyrimidine, pyrimidinyloxy(thio)benzoate orsulonylaminocarbonyltriazolinone, an acetyl Co-A carboxylase inhibitorsuch as quizalofop-P-ethyl, a synthetic auxin such as quinclorac,KIH-485 or a protox inhibitor to control the weeds without significantlydamaging the crop plants.

Generally, the effective amount of herbicide applied to the field issufficient to selectively control the weeds without significantlyaffecting the crop. “Weed” as used herein refers to a plant which is notdesirable in a particular area. Conversely, a “crop plant” as usedherein refers to a plant which is desired in a particular area, such as,for example, a Brassica plant. Thus, in some embodiments, a weed is anon-crop plant or a non-crop species, while in some embodiments, a weedis a crop species which is sought to be eliminated from a particulararea, such as, for example, an inferior and/or non-transgenic Brassicaplant in a field planted with Brassica event DP-073496-4 or anon-Brassica crop plant in a field planted with DP-073496-4. Weeds canbe classified into two major groups: monocots and dicots.

Many plant species can be controlled (i.e., killed or damaged) by theherbicides described herein. Accordingly, the methods of the inventionare useful in controlling these plant species where they are undesirable(i.e., where they are weeds). These plant species include crop plants aswell as species commonly considered weeds, including but not limited tospecies such as: blackgrass (Alopecurus myosuroides), giant foxtail(Setaria faberi), large crabgrass (Digitaria sanguinalis), Surinam grass(Brachiaria decumbens), wild oat (Avena fatua), common cocklebur(Xanthium pensylvanicum), common lambsquarters (Chenopodium album),morning glory (Ipomoea coccinea), pigweed (Amaranthus spp.), velvetleaf(Abutilion theophrasti), common barnyardgrass (Echinochloa crus-galli),bermudagrass (Cynodon dactylon), downy brome (Bromus tectorum),goosegrass (Eleusine indica), green foxtail (Setaria viridis), Italianryegrass (Lolium multiflorum), Johnsongrass (Sorghum halepense), lessercanarygrass (Phalaris minor), windgrass (Apera spica-venti), woolycupgrass (Erichloa villosa), yellow nutsedge (Cyperus esculentus),common chickweed (Stellaria media), common ragweed (Ambrosiaartemisiifolia), Kochia scoparia, horseweed (Conyza canadensis), rigidryegrass (Lolium rigidum), goosegrass (Eleucine indica), hairy fleabane(Conyza bonariensis), buckhorn plantain (Plantago lanceolata), tropicalspiderwort (Commelina benghalensis), field bindweed (Convolvulusarvensis), purple nutsedge (Cyperus rotundus), redvine (Brunnichiaovata), hemp sesbania (Sesbania exaltata), sicklepod (Sennaobtusifolia), Texas blueweed (Helianthus ciliaris) and Devil's claws(Proboscidea louisianica). In other embodiments, the weed comprises aherbicide-resistant ryegrass, for example, a glyphosate resistantryegrass, a paraquat resistant ryegrass, a ACCase-inhibitor resistantryegrass and a non-selective herbicide resistant ryegrass. In someembodiments, the undesired plants are proximate the crop plants.

As used herein, by “selectively controlled” it is intended that themajority of weeds in an area of cultivation are significantly damaged orkilled, while if crop plants are also present in the field, the majorityof the crop plants are not significantly damaged. Thus, a method isconsidered to selectively control weeds when at least 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95% or more of the weeds are significantlydamaged or killed, while if crop plants are also present in the field,less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the cropplants are significantly damaged or killed.

In some embodiments, a Brassica DP-073496-4 plant of the invention isnot significantly damaged by treatment with a particular herbicideapplied to that plant at a dose equivalent to a rate of at least 0.5, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45,50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 150, 170, 200,300, 400, 500, 600, 700, 800, 800, 1000, 2000, 3000, 4000, 5000 or moregrams or ounces (1 ounce=29.57 ml) of active ingredient or commercialproduct or herbicide formulation per acre or per hectare, whereas anappropriate control plant is significantly damaged by the sametreatment.

In specific embodiments, an effective amount of an ALS inhibitorherbicide comprises at least about 0.1, 1, 5, 10, 25, 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 600, 700, 750, 800, 850, 900, 950,1000, 2000, 3000, 4000, 5000 or more grams or ounces (1 ounce=29.57 ml)of active ingredient per hectare. In other embodiments, an effectiveamount of an ALS inhibitor comprises at least about 0.1-50, about 25-75,about 50-100, about 100-110, about 110-120, about 120-130, about130-140, about 140-150, about 150-200, about 200-500, about 500-600,about 600-800, about 800-1000 or greater grams or ounces (1 ounce=29.57ml) of active ingredient per hectare. Any ALS inhibitor, for example,those listed in Table 1 can be applied at these levels.

In other embodiments, an effective amount of a sulfonylurea comprises atleast 0.1, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 600, 700, 800, 900, 1000, 5000 or more grams or ounces (1ounce=29.57 ml) of active ingredient per hectare. In other embodiments,an effective amount of a sulfonylurea comprises at least about 0.1-50,about 25-75, about 50-100, about 100-110, about 110-120, about 120-130,about 130-140, about 140-150, about 150-160, about 160-170, about170-180, about 190-200, about 200-250, about 250-300, about 300-350,about 350-400, about 400-450, about 450-500, about 500-550, about550-600, about 600-650, about 650-700, about 700-800, about 800-900,about 900-1000, about 1000-2000 or more grams or ounces (1 ounce=29.57ml) of active ingredient per hectare. Representative sulfonylureas thatcan be applied at this level are set forth in Table 1.

In other embodiments, an effective amount of asulfonylaminocarbonyltriazolinones, triazolopyrimidines,pyrimidinyloxy(thio)benzoates, and imidazolinones can comprise at leastabout 0.1, 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450,500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100,1150, 1200, 1250, 1300, 1350, 1400, 1500, 1550, 1600, 1650, 1700, 1800,1850, 1900, 1950, 2000, 2500, 3500, 4000, 4500, 5000 or greater grams orounces (1 ounce=29.57 ml) active ingredient per hectare. In otherembodiments, an effective amount of a sulfonyluminocarbonyltriazolines,triazolopyrimidines, pyrimidinyloxy(thio)benzoates or imidazolinonescomprises at least about 0.1-50, about 25-75, about 50-100, about100-110, about 110-120, about 120-130, about 130-140, about 140-150,about 150-160, about 160-170, about 170-180, about 190-200, about200-250, about 250-300, about 300-350, about 350-400, about 400-450,about 450-500, about 500-550, about 550-600, about 600-650, about650-700, about 700-800, about 800-900, about 900-1000, about 1000-2000or more grams or ounces (1 ounce=29.57 ml) active ingredient perhectare.

Additional ranges of the effective amounts of herbicides can be found,for example, in various publications from University Extension services.See, for example, Bernards, et al., (2006) Guide for Weed Management inNebraska (www.ianrpubs.url.edu/sendlt/ec130); Regher, et al., (2005)Chemical Weed Control for Fields Crops, Pastures, Rangeland, andNoncropland, Kansas State University Agricultural Extension Station andCorporate Extension Service; Zollinger, et al., (2006) North Dakota WeedControl Guide, North Dakota Extension Service and the Iowa StateUniversity Extension at www.weeds.iastate.edu, each of which is hereinincorporated by reference.

In some embodiments of the invention, glyphosate is applied to an areaof cultivation and/or to at least one plant in an area of cultivation atrates between 8 and 32 ounces of acid equivalent per acre, or at ratesbetween 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 ounces of acidequivalent per acre at the lower end of the range of application andbetween 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32 ounces of acidequivalent per acre at the higher end of the range of application (1ounce=29.57 ml). In other embodiments, glyphosate is applied at least at1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or greater ounce of activeingredient per hectare (1 ounce=29.57 ml). In some embodiments of theinvention, a sulfonylurea herbicide is applied to a field and/or to atleast one plant in a field at rates between 0.04 and 1.0 ounces ofactive ingredient per acre, or at rates between 0.1, 0.2, 0.4, 0.6 and0.8 ounces of active ingredient per acre at the lower end of the rangeof application and between 0.2, 0.4, 0.6, 0.8 and 1.0 ounces of activeingredient per acre at the higher end of the range of application. (1ounce=29.57 ml).

As is known in the art, glyphosate herbicides as a class contain thesame active ingredient, but the active ingredient is present as one of anumber of different salts and/or formulations. However, herbicides knownto inhibit ALS vary in their active ingredient as well as their chemicalformulations. One of skill in the art is familiar with the determinationof the amount of active ingredient and/or acid equivalent present in aparticular volume and/or weight of herbicide preparation.

In some embodiments, an ALS inhibitor herbicide is employed. Rates atwhich the ALS inhibitor herbicide is applied to the crop, crop part,seed or area of cultivation can be any of the rates disclosed herein. Inspecific embodiments, the rate for the ALS inhibitor herbicide is about0.1 to about 5000 g ai/hectare, about 0.5 to about 300 g ai/hectare orabout 1 to about 150 g ai/hectare.

Generally, a particular herbicide is applied to a particular field (andany plants growing in it) no more than 1, 2, 3, 4, 5, 6, 7 or 8 times ayear, or no more than 1, 2, 3, 4 or 5 times per growing season.

By “treated with a combination of” or “applying a combination of”herbicides to a crop, area of cultivation or field” it is intended thata particular field, crop or weed is treated with each of the herbicidesand/or chemicals indicated to be part of the combination so that adesired effect is achieved, i.e., so that weeds are selectivelycontrolled while the crop is not significantly damaged. In someembodiments, weeds which are susceptible to each of the herbicidesexhibit damage from treatment with each of the herbicides which isadditive or synergistic. The application of each herbicide and/orchemical may be simultaneous or the applications may be at differenttimes, so long as the desired effect is achieved. Furthermore, theapplication can occur prior to the planting of the crop.

The proportions of herbicides used in the methods of the invention withother herbicidal active ingredients in herbicidal compositions aregenerally in the ratio of 5000:1 to 1:5000, 1000:1 to 1:1000, 100:1 to1:100, 10:1 to 1:10 or 5:1 to 1:5 by weight. The optimum ratios can beeasily determined by those skilled in the art based on the weed controlspectrum desired. Moreover, any combinations of ranges of the variousherbicides disclosed in Table 1 can also be applied in the methods ofthe invention.

Thus, in some embodiments, the invention provides improved methods forselectively controlling weeds in a field wherein the total herbicideapplication may be less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%,50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of that used inother methods. Similarly, in some embodiments, the amount of aparticular herbicide used for selectively controlling weeds in a fieldmay be less than 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the amount of that particularherbicide that would be used in other methods, i.e., methods notutilizing a plant of the invention.

In some embodiments, a DP-073496-4 Brassica plant of the inventionbenefits from a synergistic effect, wherein the herbicide toleranceconferred by the GLYAT polypeptide and that conferred by a polypeptideproviding tolerance to another herbicide is greater than expected fromsimply combining the herbicide tolerance conferred by each geneseparately. See, e.g., McCutchen, et al., (1997) J. Econ. Entomol.90:1170-1180; Priesler, et al., (1999) J. Econ. Entomol. 92:598-603. Asused herein, the terms “synergy,” “synergistic,” “synergistically” andderivations thereof, such as in a “synergistic effect” or a “synergisticherbicide combination” or a “synergistic herbicide composition” refer tocircumstances under which the biological activity of a combination ofherbicides, such as at least a first herbicide and a second herbicide,is greater than the sum of the biological activities of the individualherbicides. Synergy, expressed in terms of a “Synergy Index (SI),”generally can be determined by the method described by Kull, et al.,(1961) Applied Microbiology 9:538. See also, Colby, (1967) Weeds15:20-22.

In other instances, the herbicide tolerance conferred on a DP-073496-4plant of the invention is additive; that is, the herbicide toleranceprofile conferred by the herbicide tolerance genes is what would beexpected from simply combining the herbicide tolerance conferred by eachgene separately to a transgenic plant containing them individually.Additive and/or synergistic activity for two or more herbicides againstkey weed species will increase the overall effectiveness and/or reducethe actual amount of active ingredient(s) needed to control said weeds.Where such synergy is observed, the plant of the invention may displaytolerance to a higher dose or rate of herbicide and/or the plant maydisplay tolerance to additional herbicides or other chemicals beyondthose to which it would be expected to display tolerance. For example, aDP-073496-4 Brassica plant may show tolerance to organophosphatecompounds such as insecticides and/or inhibitors of4-hydroxyphenylpyruvate dioxygenase.

Thus, for example, the DP-073496-4 Brassica plants of the invention,when further comprising genes conferring tolerance to other herbicides,can exhibit greater than expected tolerance to various herbicides,including but not limited to glyphosate, ALS inhibitor chemistries andsulfonylurea herbicides. The DP-073496-4 Brassica plant plants of theinvention may show tolerance to a particular herbicide or herbicidecombination that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,12%, 15%, 17%, 20%, 22%, 25%, 27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 300%, 400% or 500% ormore higher than the tolerance of an appropriate control plant thatcontains only a single herbicide tolerance gene which confers toleranceto the same herbicide or herbicide combination. Thus, DP-073496-4Brassica plants may show decreased damage from the same dose ofherbicide in comparison to an appropriate control plant, or they mayshow the same degree of damage in response to a much higher dose ofherbicide than the control plant. Accordingly, in specific embodiments,a particular herbicide used for selectively containing weeds in a fieldis more than 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%,70%, 80%, 90%, 100% or greater than the amount of that particularherbicide that would be used in other methods, i.e., methods notutilizing a plant of the invention.

In the same manner, in some embodiments, a DP-073496-4 Brassica plant ofthe invention shows improved tolerance to a particular formulation of aherbicide active ingredient in comparison to an appropriate controlplant. Herbicides are sold commercially as formulations which typicallyinclude other ingredients in addition to the herbicide activeingredient; these ingredients are often intended to enhance the efficacyof the active ingredient. Such other ingredients can include, forexample, safeners and adjuvants (see, e.g., Green and Foy, (2003)“Adjuvants: Tools for Enhancing Herbicide Performance,” in Weed Biologyand Management, ed. Inderjit (Kluwer Academic Publishers, TheNetherlands)). Thus, a DP-073496-4 Brassica plant of the invention canshow tolerance to a particular formulation of a herbicide (e.g., aparticular commercially available herbicide product) that is at least1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 15%, 17%, 20%, 22%, 25%,27%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 80%, 90%, 100%, 125%,150%, 175%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000%,1100%, 1200%, 1300%, 1400%, 1500%, 1600%, 1700%, 1800%, 1900% or 2000%or more higher than the tolerance of an appropriate control plant thatcontains only a single herbicide tolerance gene which confers toleranceto the same herbicide formulation.

In some embodiments, a DP-073496-4 Brassica plant of the invention showsimproved tolerance to a herbicide or herbicide class to which at leastone other herbicide tolerance gene confers tolerance as well as improvedtolerance to at least one other herbicide or chemical which has adifferent mechanism or basis of action than either glyphosate or theherbicide corresponding to said at least one other herbicide tolerancegene. This surprising benefit of the invention finds use in methods ofgrowing crops that comprise treatment with various combinations ofchemicals, including, for example, other chemicals used for growingcrops. Thus, for example, a DP-073496-4 Brassica plant may also showimproved tolerance to chlorpyrifos, a systemic organophosphateinsecticide. Thus, the invention also provides a DP-073496-4 Brassicaplant that confers tolerance to glyphosate (i.e., a GLYAT gene) whichshows improved tolerance to chemicals which affect the cytochrome P450gene, and methods of use thereof. In some embodiments, the DP-073496-4Brassica plants also show improved tolerance to dicamba. In theseembodiments, the improved tolerance to dicamba may be evident in thepresence of glyphosate and a sulfonylurea herbicide.

In other methods, a herbicide combination is applied over a DP-073496-4Brassica plant, where the herbicide combination produces either anadditive or a synergistic effect for controlling weeds. Suchcombinations of herbicides can allow the application rate to be reduced,a broader spectrum of undesired vegetation to be controlled, improvedcontrol of the undesired vegetation with fewer applications, more rapidonset of the herbicidal activity or more prolonged herbicidal activity.

An “additive herbicidal composition” has a herbicidal activity that isabout equal to the observed activities of the individual components. A“synergistic herbicidal combination” has a herbicidal activity higherthan what can be expected based on the observed activities of theindividual components when used alone. Accordingly, the presentlydisclosed subject matter provides a synergistic herbicide combination,wherein the degree of weed control of the mixture exceeds the sum ofcontrol of the individual herbicides. In some embodiments, the degree ofweed control of the mixture exceeds the sum of control of the individualherbicides by any statistically significant amount including, forexample, about 1% to 5%, about 5% to about 10%, about 10% to about 20%,about 20% to about 30%, about 30% to 40%, about 40% to about 50%, about50% to about 60%, about 60% to about 70%, about 70% to about 80%, about80% to about 90%, about 90% to about 100%, about 100% to 120% orgreater. Further, a “synergistically effective amount” of a herbiciderefers to the amount of one herbicide necessary to elicit a synergisticeffect in another herbicide present in the herbicide composition. Thus,the term “synergist,” and derivations thereof, refer to a substance thatenhances the activity of an active ingredient (ai), i.e., a substance ina formulation from which a biological effect is obtained, for example, aherbicide.

Accordingly, in some embodiments, the presently disclosed subject matterprovides a method for controlling weeds in an area of cultivation. Insome embodiments, the method comprises: (a) planting the area with aDP-073496-4 crop seeds or crop plants which also comprisepolynucleotides conferring ALS-inhibitor tolerance; and (b) applying tothe weed, the crop plants, a crop part, the area of cultivation or acombination thereof, an effective amount of a herbicide compositioncomprising at least one of a synergistically effective amount ofglyphosate and a synergistically effective amount of an ALS inhibitor(for example, but not limited to, a sulfonylurea herbicide) oragriculturally suitable salts thereof, wherein at least one of: (i) thesynergistically effective amount of the glyphosate is lower than anamount of glyphosate required to control the weeds in the absence of thesulfonylurea herbicide; (ii) the synergistically effective amount of theALS inhibitor herbicide is lower than an amount of the ALS inhibitorrequired to control the weeds in the absence of glyphosate and (iii)combinations thereof and wherein the effective amount of the herbicidecomposition is tolerated by the crop seeds or crop plants and controlsthe weeds in the area of cultivation.

In some embodiments, the herbicide composition used in the presentlydisclosed method for controlling weeds comprises a synergisticallyeffective amount of glyphosate and a sulfonylurea herbicide. In furtherembodiments, the presently disclosed synergistic herbicide compositioncomprises glyphosate and a sulfonylurea herbicide selected from thegroup consisting of metsulfuron-methyl, chlorsulfuron and triasulfuron.

In particular embodiments, the synergistic herbicide combination furthercomprises an adjuvant such as, for example, an ammonium sulfate-basedadjuvant, e.g., ADD-UP® (Wenkem., Halfway House, Midrand, South Africa).In additional embodiments, the presently disclosed synergistic herbicidecompositions comprise an additional herbicide, for example, an effectiveamount of a pyrimidinyloxy(thio)benzoate herbicide. In some embodiments,the pyrimidinyloxy(thio)benzoate herbicide comprises bispyribac, e.g.,(VELOCITY®, Valent U.S.A. Corp., Walnut Creek, Calif., United States ofAmerica) or an agriculturally suitable salt thereof.

In some embodiments of the presently disclosed method for controllingundesired plants, the glyphosate is applied pre-emergence,post-emergence or pre- and post-emergence to the undesired plants orplant crops and/or the ALS inhibitor herbicide (i.e., the sulfonylureaherbicide) is applied pre-emergence, post-emergence or pre- andpost-emergence to the undesired plants or plant crops. In otherembodiments, the glyphosate and/or the ALS inhibitor herbicide (i.e.,the sulfonylurea herbicide) are applied together or are appliedseparately. In yet other embodiments, the synergistic herbicidecomposition is applied, e.g., step (b) above, at least once prior toplanting the crop(s) of interest, e.g., step (a) above.

Weeds that can be difficult to control with glyphosate alone in fieldswhere a crop is grown (such as, for example, a brassica crop) includebut are not limited to the following: horseweed (e.g., Conyzacanadensis); rigid ryegrass (e.g., Lolium rigidum); goosegrass (e.g.,Eleusine indica); Italian ryegrass (e.g., Lolium multiflorum); hairyfleabane (e.g., Conyza bonariensis); buckhorn plantain (e.g., Plantagolanceolate); common ragweed (e.g., Ambrosia artemisifolia); morningglory (e.g., Ipomoea spp.); waterhemp (e.g., Amaranthus spp.); fieldbindweed (e.g., Convolvulus arvensis); yellow nutsedge (e.g., Cyperusesculentus); common lambsquarters (e.g., Chenopodium album); wildbuckwheat (e.g., Polygonium convolvulus); velvetleaf (e.g., Abutilontheophrasti); kochia (e.g., Kochia scoparia) and Asiatic dayflower(e.g., Commelina spp.). In areas where such weeds are found, Brassicaplants comprising the DP-073496-4 event and tolerance to anotherherbicide are particularly useful in allowing the treatment of a field(and therefore any crop growing in the field) with combinations ofherbicides that would cause unacceptable damage to crop plants that didnot contain both of these polynucleotides. Plants of the invention thatare tolerant to glyphosate and other herbicides such as, for example,sulfonylurea, imidazolinone, triazolopyrimidine,pyrimidinyl(thio)benzoate and/or sulfonylaminocarbonyltriazolinoneherbicides in addition to being tolerant to at least one other herbicidewith a different mode of action or site of action are particularlyuseful in situations where weeds are tolerant to at least two of thesame herbicides to which the plants are tolerant. In this manner, plantsof the invention make possible improved control of weeds that aretolerant to more than one herbicide.

For example, some commonly used treatments for weed control in fieldswhere current commercial crops (including, for example, Brassicas) aregrown include glyphosate and, optionally, 2,4-D; this combination,however, has some disadvantages. Particularly, there are weed speciesthat it does not control well and it also does not work well for weedcontrol in cold weather. Another commonly used treatment for weedcontrol in brassica fields is the sulfonylurea herbicidechlorimuron-ethyl, which has significant residual activity in the soiland thus maintains selective pressure on all later-emerging weedspecies, creating a favorable environment for the growth and spread ofsulfonylurea-resistant weeds. Fields may be treated with sulfonylurea,imidazolinone, triazolopyrimidines, pyrimidiny(thio)benzoates and/orsulfonylaminocarbonyltriazonlinone such as the sulfonylureachlorimuron-ethyl, either alone or in combination with other herbicides,such as a combination of glyphosate and tribenuron-methyl (availablecommercially as Express®). This combination has several advantages forweed control under some circumstances, including the use of herbicideswith different modes of action and the use of herbicides having arelatively short period of residual activity in the soil. A herbicidehaving a relatively short period of residual activity is desirable, forexample, in situations where it is important to reduce selectivepressure that would favor the growth of herbicide-tolerant weeds. Ofcourse, in any particular situation where weed control is required,other considerations may be more important, such as, for example, theneed to prevent the development of and/or appearance of weeds in a fieldprior to planting a crop by using a herbicide with a relatively longperiod of residual activity. Treatments that include bothtribenuron-methyl and thifensulfuron-methyl may be particularly useful.

Other commonly used treatments for weed control in fields where currentcommercial varieties of crops (including, for example, Brassicas) aregrown include the sulfonylurea herbicide thifensulfuron-methyl(available commercially as Harmony GT®). However, one disadvantage ofthifensulfuron-methyl is that the higher application rates required forconsistent weed control often cause injury to a crop growing in the samefield. DP-073496-4 Brassica plants comprising additional tolerance canbe treated with a combination of glyphosate and thifensulfuron-methyl,which has the advantage of using herbicides with different modes ofaction. Thus, weeds that are resistant to either herbicide alone arecontrolled by the combination of the two herbicides, and the improvedDP-073496-4 Brassica plants would not be significantly damaged by thetreatment.

Other herbicides which are used for weed control in fields where currentcommercial varieties of crops (including, for example, Brassicas) aregrown are the triazolopyrimidine herbicide cloransulam-methyl (availablecommercially as FirstRate®) and the imidazolinone herbicide imazaquin(available commercially as Sceptor®). When these herbicides are usedindividually they may provide only marginal control of weeds. However,may be treated, for example, with a combination of glyphosate (e.g.,Roundup® (glyphosate isopropylamine salt)), imazapyr (currentlyavailable commercially as Arsenal®), chlorimuron-ethyl (currentlyavailable commercially as Classic®), quizalofop-P-ethyl (currentlyavailable commercially as Assure II®) and fomesafen (currently availablecommercially as Flexstar®). This combination has the advantage of usingherbicides with different modes of action. Thus, weeds that are tolerantto just one or several of these herbicides are controlled by thecombination of the five herbicides. This combination provides anextremely broad spectrum of protection against the type ofherbicide-tolerant weeds that might be expected to arise and spreadunder current weed control practices.

Fields containing the DP-073496-4 Brassica plants with additionalherbicide tolerance may also be treated, for example, with a combinationof herbicides including glyphosate, rimsulfuron, and dicamba ormesotrione. This combination may be particularly useful in controllingweeds which have developed some tolerance to herbicides which inhibitALS. Another combination of herbicides which may be particularly usefulfor weed control includes glyphosate and at least one of the following:metsulfuron-methyl (commercially available as Ally®), imazapyr(commercially available as Arsenal®), imazethapyr, imazaquin andsulfentrazone. It is understood that any of the combinations discussedabove or elsewhere herein may also be used to treat areas in combinationwith any other herbicide or agricultural chemical.

Some commonly-used treatments for weed control in fields where currentcommercial crops (including, for example, Brassica) are grown includeglyphosate (currently available commercially as Roundup®), rimsulfuron(currently available commercially as Resolve® or Matrix®), dicamba(commercially available as Clarity®), atrazine and mesotrione(commercially available as Callisto®). These herbicides are sometimesused individually due to poor crop tolerance to multiple herbicides.Unfortunately, when used individually, each of these herbicides hassignificant disadvantages. Particularly, the incidence of weeds that aretolerant to individual herbicides continues to increase, renderingglyphosate less effective than desired in some situations. Rimsulfuronprovides better weed control at high doses which can cause injury to acrop, and alternatives such as dicamba are often more expensive thanother commonly-used herbicides

Some commonly-used treatments for weed control in fields where currentcommercial crops are grown include glyphosate (currently availablecommercially as Roundup®), chlorimuron-ethyl, tribenuron-methyl,rimsulfuron (currently available commercially as Resolve® or Matrix®),imazethapyr, imazapyr and imazaquin. Unfortunately, when usedindividually, each of these herbicides has significant disadvantages.Particularly, the incidence of weeds that are tolerant to individualherbicides continues to increase, rendering each individual herbicideless effective than desired in some situations. However, DP-073496-4Brassica with an additional herbicide tolerance trait can be treatedwith a combination of herbicides that would cause unacceptable damage tostandard plant varieties, including combinations of herbicides thatinclude at least one of those mentioned above.

In the methods of the invention, a herbicide may be formulated andapplied to an area of interest such as, for example, a field or area ofcultivation, in any suitable manner. A herbicide may be applied to afield in any form, such as, for example, in a liquid spray or as solidpowder or granules. In specific embodiments, the herbicide orcombination of herbicides that are employed in the methods comprise atankmix or a premix. A herbicide may also be formulated, for example, asa “homogenous granule blend” produced using blends technology (see,e.g., U.S. Pat. No. 6,022,552, entitled “Uniform Mixtures of PesticideGranules”). The blends technology of U.S. Pat. No. 6,022,552 produces anonsegregating blend (i.e., a “homogenous granule blend”) of formulatedcrop protection chemicals in a dry granule form that enables delivery ofcustomized mixtures designed to solve specific problems. A homogenousgranule blend can be shipped, handled, subsampled and applied in thesame manner as traditional premix products where multiple activeingredients are formulated into the same granule.

Briefly, a “homogenous granule blend” is prepared by mixing together atleast two extruded formulated granule products. In some embodiments,each granule product comprises a registered formulation containing asingle active ingredient which is, for example, a herbicide, a fungicideand/or an insecticide. The uniformity (homogeneity) of a “homogenousgranule blend” can be optimized by controlling the relative sizes andsize distributions of the granules used in the blend. The diameter ofextruded granules is controlled by the size of the holes in the extruderdie and a centrifugal sifting process may be used to obtain a populationof extruded granules with a desired length distribution (see, e.g., U.S.Pat. No. 6,270,025).

A homogenous granule blend is considered to be “homogenous” when it canbe subsampled into appropriately sized aliquots and the composition ofeach aliquot will meet the required assay specifications. To demonstratehomogeneity, a large sample of the homogenous granule blend is preparedand is then subsampled into aliquots of greater than the minimumstatistical sample size. Blends also afford the ability to add otheragrochemicals at normal, labeled use rates such as additional herbicides(a 3^(rd)/4^(th) mechanism of action), fungicides, insecticides, plantgrowth regulators and the like thereby saving costs associated withadditional applications.

Any herbicide formulation applied over the DP-073496-4 Brassica plantcan be prepared as a “tank-mix” composition. In such embodiments, eachingredient or a combination of ingredients can be stored separately fromone another. The ingredients can then be mixed with one another prior toapplication. Typically, such mixing occurs shortly before application.In a tank-mix process, each ingredient, before mixing, typically ispresent in water or a suitable organic solvent. For additional guidanceregarding the art of formulation, see, Woods, “The Formulator'sToolbox—Product Forms for Modern Agriculture” Pesticide Chemistry andBioscience, The Food-Environment Challenge, Brooks and Roberts, Eds.,Proceedings of the 9th International Congress on Pesticide Chemistry,The Royal Society of Chemistry, Cambridge, 1999, pp. 120-133. See also,U.S. Pat. No. 3,235,361, Column 6, line 16 through Column 7, line 19 andExamples 10-41; U.S. Pat. No. 3,309,192, Column 5, line 43 throughColumn 7, line 62 and Examples 8, 12, 15, 39, 41, 52, 53, 58, 132,138-140, 162-164, 166, 167 and 169-182; U.S. Pat. No. 2,891,855, Column3, line 66 through Column 5, line 17 and Examples 1-4; Klingman, WeedControl as a Science, John Wiley and Sons, Inc., New York, 1961, pp81-96 and Hance, et al., Weed Control Handbook, 8th Ed., BlackwellScientific Publications, Oxford, 1989, each of which is incorporatedherein by reference in their entirety.

The methods of the invention further allow for the development ofherbicide combinations to be used with the DP-073496-4 Brassica plants.In such methods, the environmental conditions in an area of cultivationare evaluated. Environmental conditions that can be evaluated include,but are not limited to, ground and surface water pollution concerns,intended use of the crop, crop tolerance, soil residuals, weeds presentin area of cultivation, soil texture, pH of soil, amount of organicmatter in soil, application equipment and tillage practices. Upon theevaluation of the environmental conditions, an effective amount of acombination of herbicides can be applied to the crop, crop part, seed ofthe crop or area of cultivation.

In some embodiments, the herbicide applied to the DP-073496-4 Brassicaplants of the invention serves to prevent the initiation of growth ofsusceptible weeds and/or serve to cause damage to weeds that are growingin the area of interest. In some embodiments, the herbicide or herbicidemixture exert these effects on weeds affecting crops that aresubsequently planted in the area of interest (i.e., field or area ofcultivation). In the methods of the invention, the application of theherbicide combination need not occur at the same time. So long as thefield in which the crop is planted contains detectable amounts of thefirst herbicide and the second herbicide is applied at some time duringthe period in which the crop is in the area of cultivation, the crop isconsidered to have been treated with a mixture of herbicides accordingto the invention. Thus, methods of the invention encompass applicationsof herbicide which are “preemergent,” “postemergent,” “preplantincorporation” and/or which involve seed treatment prior to planting.

In one embodiment, methods are provided for coating seeds. The methodscomprise coating a seed with an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein). The seeds canthen be planted in an area of cultivation. Further provided are seedshaving a coating comprising an effective amount of a herbicide or acombination of herbicides (as disclosed elsewhere herein).

“Preemergent” refers to a herbicide which is applied to an area ofinterest (e.g., a field or area of cultivation) before a plant emergesvisibly from the soil. “Postemergent” refers to a herbicide which isapplied to an area after a plant emerges visibly from the soil. In someinstances, the terms “preemergent” and “postemergent” are used withreference to a weed in an area of interest, and in some instances theseterms are used with reference to a crop plant in an area of interest.When used with reference to a weed, these terms may apply to only aparticular type of weed or species of weed that is present or believedto be present in the area of interest. While any herbicide may beapplied in a preemergent and/or postemergent treatment, some herbicidesare known to be more effective in controlling a weed or weeds whenapplied either preemergence or postemergence. For example, rimsulfuronhas both preemergence and postemergence activity, while other herbicideshave predominately preemergence (metolachlor) or postemergence(glyphosate) activity. These properties of particular herbicides areknown in the art and are readily determined by one of skill in the art.Further, one of skill in the art would readily be able to selectappropriate herbicides and application times for use with the transgenicplants of the invention and/or on areas in which transgenic plants ofthe invention are to be planted. “Preplant incorporation” involves theincorporation of compounds into the soil prior to planting.

Thus, the invention provides improved methods of growing a crop and/orcontrolling weeds such as, for example, “pre-planting burn down,”wherein an area is treated with herbicides prior to planting the crop ofinterest in order to better control weeds. The invention also providesmethods of growing a crop and/or controlling weeds which are “no-till”or “low-till” (also referred to as “reduced tillage”). In such methods,the soil is not cultivated or is cultivated less frequently during thegrowing cycle in comparison to traditional methods; these methods cansave costs that would otherwise be incurred due to additionalcultivation, including labor and fuel costs.

The methods of the invention encompass the use of simultaneous and/orsequential applications of multiple classes of herbicides. In someembodiments, the methods of the invention involve treating a plant ofthe invention and/or an area of interest (e.g., a field or area ofcultivation) and/or weed with just one herbicide or other chemical suchas, for example, a sulfonylurea herbicide.

The time at which a herbicide is applied to an area of interest (and anyplants therein) may be important in optimizing weed control. The time atwhich a herbicide is applied may be determined with reference to thesize of plants and/or the stage of growth and/or development of plantsin the area of interest, e.g., crop plants or weeds growing in the area.The stages of growth and/or development of plants are known in the art.Thus, for example, the time at which a herbicide or other chemical isapplied to an area of interest in which plants are growing may be thetime at which some or all of the plants in a particular area havereached at least a particular size and/or stage of growth and/ordevelopment, or the time at which some or all of the plants in aparticular area have not yet reached a particular size and/or stage ofgrowth and/or development.

In some embodiments, the DP-073496-4 Brassica plants of the inventionshow improved tolerance to postemergence herbicide treatments. Forexample, plants of the invention may be tolerant to higher doses ofherbicide, tolerant to a broader range of herbicides, and/or may betolerant to doses of herbicide applied at earlier or later times ofdevelopment in comparison to an appropriate control plant. For example,in some embodiments, the DP-073496-4 Brassica plants of the inventionshow an increased resistance to morphological defects that are known toresult from treatment at particular stages of development. Thus, theglyphosate-tolerant plants of the invention find use in methodsinvolving herbicide treatments at later stages of development than werepreviously feasible. Thus, plants of the invention may be treated with aparticular herbicide that causes morphological defects in a controlplant treated at the same stage of development, but theglyphosate-tolerant plants of the invention will not be significantlydamaged by the same treatment.

Different chemicals such as herbicides have different “residual”effects, i.e., different amounts of time for which treatment with thechemical or herbicide continues to have an effect on plants growing inthe treated area. Such effects may be desirable or undesirable,depending on the desired future purpose of the treated area (e.g., fieldor area of cultivation). Thus, a crop rotation scheme may be chosenbased on residual effects from treatments that will be used for eachcrop and their effect on the crop that will subsequently be grown in thesame area. One of skill in the art is familiar with techniques that canbe used to evaluate the residual effect of a herbicide; for example,generally, glyphosate has very little or no soil residual activity,while herbicides that act to inhibit ALS vary in their residual activitylevels. Residual activities for various herbicides are known in the art,and are also known to vary with various environmental factors such as,for example, soil moisture levels, temperature, pH and soil composition(texture and organic matter).

Moreover, the transgenic plants of the invention provide improvedtolerance to treatment with additional chemicals commonly used on cropsin conjunction with herbicide treatments, such as safeners, adjuvantssuch as ammonium sulfonate and crop oil concentrate, and the like. Theterm “safener” refers to a substance that when added to a herbicideformulation eliminates or reduces the phytotoxic effects of theherbicide to certain crops. One of ordinary skill in the art wouldappreciate that the choice of safener depends, in part, on the cropplant of interest and the particular herbicide or combination ofherbicides included in the synergistic herbicide composition. Exemplarysafeners suitable for use with the presently disclosed herbicidecompositions include, but are not limited to, those disclosed in U.S.Pat. Nos. 4,808,208; 5,502,025; 6,124,240 and US Patent ApplicationPublication Numbers 2006/0148647; 2006/0030485; 2005/0233904;2005/0049145; 2004/0224849; 2004/0224848; 2004/0224844; 2004/0157737;2004/0018940; 2003/0171220; 2003/0130120; 2003/0078167, the disclosuresof which are incorporated herein by reference in their entirety. Themethods of the invention can involve the use of herbicides incombination with herbicide safeners such as benoxacor, BCS(1-bromo-4-[(chloromethyl)sulfonyl]benzene), cloquintocet-mexyl,cyometrinil, dichlormid, 2-(dichloromethyl)-2-methyl-1,3-dioxolane (MG191), fenchlorazole-ethyl, fenclorim, flurazole, fluxofenim, furilazole,isoxadifen-ethyl, mefenpyr-diethyl, methoxyphenone((4-methoxy-3-methylphenyl)(3-methylphenyl)-methanone), naphthalicanhydride (1,8-naphthalic anhydride) and oxabetrinil to increase cropsafety. Antidotally effective amounts of the herbicide safeners can beapplied at the same time as the compounds of this invention, or appliedas seed treatments. Therefore an aspect of the present invention relatesto the use of a mixture comprising glyphosate, at least one otherherbicide and an antidotally effective amount of a herbicide safener.

Seed treatment is particularly useful for selective weed control,because it physically restricts antidoting to the crop plants. Thereforea particularly useful embodiment of the present invention is a methodfor selectively controlling the growth of weeds in a field comprisingtreating the seed from which the crop is grown with an antidotallyeffective amount of safener and treating the field with an effectiveamount of herbicide to control weeds. Antidotally effective amounts ofsafeners can be easily determined by one skilled in the art throughsimple experimentation. An antidotally effective amount of a safener ispresent where a desired plant is treated with the safener so that theeffect of a herbicide on the plant is decreased in comparison to theeffect of the herbicide on a plant that was not treated with thesafener; generally, an antidotally effective amount of safener preventsdamage or severe damage to the plant treated with the safener. One ofskill in the art is capable of determining whether the use of a safeneris appropriate and determining the dose at which a safener should beadministered to a crop.

In specific embodiments, the herbicide or herbicide combination appliedto the plant of the invention acts as a safener. In this embodiment, afirst herbicide or a herbicide mixture is applied at an antidotallyeffect amount to the plant. Accordingly, a method for controlling weedsin an area of cultivation is provided. The method comprises planting thearea with crop seeds or plants which comprise a first polynucleotideencoding a polypeptide that can confer tolerance to glyphosate operablylinked to a promoter active in a plant; and, a second polynucleotideencoding an ALS inhibitor-tolerant polypeptide operably linked to apromoter active in a plant. A combination of herbicides comprising atleast an effective amount of a first and a second herbicide is appliedto the crop, crop part, weed or area of cultivation thereof. Theeffective amount of the herbicide combination controls weeds; and, theeffective amount of the first herbicide is not tolerated by the cropwhen applied alone when compared to a control crop that has not beenexposed to the first herbicide; and, the effective amount of the secondherbicide is sufficient to produce a safening effect, wherein thesafening effect provides an increase in crop tolerance upon theapplication of the first and the second herbicide when compared to thecrop tolerance when the first herbicide is applied alone.

In specific embodiments, the combination of safening herbicidescomprises a first ALS inhibitor and a second ALS inhibitor. In otherembodiments, the safening effect is achieved by applying an effectiveamount of a combination of glyphosate and at least one ALS inhibitorchemistry. Such mixtures provides increased crop tolerance (i.e., adecrease in herbicidal injury). This method allows for increasedapplication rates of the chemistries post or pre-treatment. Such methodsfind use for increased control of unwanted or undesired vegetation. Instill other embodiments, a safening affect is achieved when theDP-073496-4 brassica crops, crop part, crop seed, weed or area ofcultivation is treated with at least one herbicide from the sulfonylureafamily of chemistry in combination with at least one herbicide from theimidazolinone family. This method provides increased crop tolerance(i.e., a decrease in herbicidal injury). In specific embodiments, thesulfonylurea comprises rimsulfuron and the imidazolinone comprisesimazethapyr. In other embodiments, glyphosate is also applied to thecrop, crop part or area of cultivation.

As used herein, an “adjuvant” is any material added to a spray solutionor formulation to modify the action of an agricultural chemical or thephysical properties of the spray solution. See, for example, Green andFoy, (2003) “Adjuvants: Tools for Enhancing Herbicide Performance,” inWeed Biology and Management, ed. Inderjit (Kluwer Academic Publishers,The Netherlands). Adjuvants can be categorized or subclassified asactivators, acidifiers, buffers, additives, adherents, antiflocculants,antifoamers, defoamers, antifreezes, attractants, basic blends,chelating agents, cleaners, colorants or dyes, compatibility agents,cosolvents, couplers, crop oil concentrates, deposition agents,detergents, dispersants, drift control agents, emulsifiers, evaporationreducers, extenders, fertilizers, foam markers, formulants, inerts,humectants, methylated seed oils, high load COCs, polymers, modifiedvegetable oils, penetrators, repellants, petroleum oil concentrates,preservatives, rainfast agents, retention aids, solubilizers,surfactants, spreaders, stickers, spreader stickers, synergists,thickeners, translocation aids, uv protectants, vegetable oils, waterconditioners and wetting agents.

In addition, methods of the invention can comprise the use of aherbicide or a mixture of herbicides, as well as, one or more otherinsecticides, fungicides, nematocides, bactericides, acaricides, growthregulators, chemosterilants, semiochemicals, repellents, attractants,pheromones, feeding stimulants or other biologically active compounds orentomopathogenic bacteria, virus or fungi to form a multi-componentmixture giving an even broader spectrum of agricultural protection.Examples of such agricultural protectants which can be used in methodsof the invention include: insecticides such as abamectin, acephate,acetamiprid, amidoflumet (S-1955), avermectin, azadirachtin,azinphos-methyl, bifenthrin, bifenazate, buprofezin, carbofuran, cartap,chlorfenapyr, chlorfluazuron, chlorpyrifos, chlorpyrifos-methyl,chromafenozide, clothianidin, cyflumetofen, cyfluthrin, beta-cyfluthrin,cyhalothrin, lambda-cyhalothrin, cypermethrin, cyromazine, deltamethrin,diafenthiuron, diazinon, dieldrin, diflubenzuron, dimefluthrin,dimethoate, dinotefuran, diofenolan, emamectin, endosulfan,esfenvalerate, ethiprole, fenothiocarb, fenoxycarb, fenpropathrin,fenvalerate, fipronil, flonicamid, flubendiamide, flucythrinate,tau-fluvalinate, flufenerim (UR-50701), flufenoxuron, fonophos,halofenozide, hexaflumuron, hydramethylnon, imidacloprid, indoxacarb,isofenphos, lufenuron, malathion, metaflumizone, metaldehyde,methamidophos, methidathion, methomyl, methoprene, methoxychlor,metofluthrin, monocrotophos, methoxyfenozide, nitenpyram, nithiazine,novaluron, noviflumuron (XDE-007), oxamyl, parathion, parathion-methyl,permethrin, phorate, phosalone, phosmet, phosphamidon, pirimicarb,profenofos, profluthrin, pymetrozine, pyrafluprole, pyrethrin,pyridalyl, pyriprole, pyriproxyfen, rotenone, ryanodine, spinosad,spirodiclofen, spiromesifen (BSN 2060), spirotetramat, sulprofos,tebufenozide, teflubenzuron, tefluthrin, terbufos, tetrachlorvinphos,thiacloprid, thiamethoxam, thiodicarb, thiosultap-sodium, tralomethrin,triazamate, trichlorfon and triflumuron; fungicides such as acibenzolar,aldimorph, amisulbrom, azaconazole, azoxystrobin, benalaxyl, benomyl,benthiavalicarb, benthiavalicarb-isopropyl, binomial, biphenyl,bitertanol, blasticidin-S, Bordeaux mixture (Tribasic copper sulfate),boscalid/nicobifen, bromuconazole, bupirimate, buthiobate, carboxin,carpropamid, captafol, captan, carbendazim, chloroneb, chlorothalonil,chlozolinate, clotrimazole, copper oxychloride, copper salts such ascopper sulfate and copper hydroxide, cyazofamid, cyflunamid, cymoxanil,cyproconazole, cyprodinil, dichlofluanid, diclocymet, diclomezine,dicloran, diethofencarb, difenoconazole, dimethomorph, dimoxystrobin,diniconazole, diniconazole-M, dinocap, discostrobin, dithianon,dodemorph, dodine, econazole, etaconazole, edifenphos, epoxiconazole,ethaboxam, ethirimol, ethridiazole, famoxadone, fenamidone, fenarimol,fenbuconazole, fencaramid, fenfuram, fenhexamide, fenoxanil,fenpiclonil, fenpropidin, fenpropimorph, fentin acetate, fentinhydroxide, ferbam, ferfurazoate, ferimzone, fluazinam, fludioxonil,flumetover, fluopicolide, fluoxastrobin, fluquinconazole,fluquinconazole, flusilazole, flusulfamide, flutolanil, flutriafol,folpet, fosetyl-aluminum, fuberidazole, furalaxyl, furametapyr,hexaconazole, hymexazole, guazatine, imazalil, imibenconazole,iminoctadine, iodicarb, ipconazole, iprobenfos, iprodione, iprovalicarb,isoconazole, isoprothiolane, kasugamycin, kresoxim-methyl, mancozeb,mandipropamid, maneb, mapanipyrin, mefenoxam, mepronil, metalaxyl,metconazole, methasulfocarb, metiram, metominostrobin/fenominostrobin,mepanipyrim, metrafenone, miconazole, myclobutanil, neo-asozin (ferricmethanearsonate), nuarimol, octhilinone, ofurace, orysastrobin,oxadixyl, oxolinic acid, oxpoconazole, oxycarboxin, paclobutrazol,penconazole, pencycuron, penthiopyrad, perfurazoate, phosphonic acid,phthalide, picobenzamid, picoxystrobin, polyoxin, probenazole,prochloraz, procymidone, propamocarb, propamocarb-hydrochloride,propiconazole, propineb, proquinazid, prothioconazole, pyraclostrobin,pryazophos, pyrifenox, pyrimethanil, pyrifenox, pyroInitrine,pyroquilon, quinconazole, quinoxyfen, quintozene, silthiofam,simeconazole, spiroxamine, streptomycin, sulfur, tebuconazole,techrazene, tecloftalam, tecnazene, tetraconazole, thiabendazole,thifluzamide, thiophanate, thiophanate-methyl, thiram, tiadinil,tolclofos-methyl, tolyfluanid, triadimefon, triadimenol, triarimol,triazoxide, tridemorph, trimoprhamide tricyclazole, trifloxystrobin,triforine, triticonazole, uniconazole, validamycin, vinclozolin, zineb,ziram, and zoxamide; nematocides such as aldicarb, oxamyl andfenamiphos; bactericides such as streptomycin; acaricides such asamitraz, chinomethionat, chlorobenzilate, cyhexatin, dicofol,dienochlor, etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin,fenpyroximate, hexythiazox, propargite, pyridaben and tebufenpyrad andbiological agents including entomopathogenic bacteria, such as Bacillusthuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki,and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g.,Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardinefungus; and entomopathogenic virus including baculovirus,nucleopolyhedro virus (NPV) such as HzNPV, AfNPV; and granulosis virus(GV) such as CpGV. The weight ratios of these various mixing partners toother compositions (e.g., herbicides) used in the methods of theinvention typically are between 100:1 and 1:100, or between 30:1 and1:30, between 10:1 and 1:10, or between 4:1 and 1:4.

The present invention also pertains to a composition comprising abiologically effective amount of a herbicide of interest or a mixture ofherbicides, and an effective amount of at least one additionalbiologically active compound or agent and can further comprise at leastone of a surfactant, a solid diluent or a liquid diluent. Examples ofsuch biologically active compounds or agents are: insecticides such asabamectin, acephate, acetamiprid, amidoflumet (S-1955), avermectin,azadirachtin, azinphos-methyl, bifenthrin, binfenazate, buprofezin,carbofuran, chlorfenapyr, chlorfluazuron, chlorpyrifos,chlorpyrifos-methyl, chromafenozide, clothianidin, cyfluthrin,beta-cyfluthrin, cyhalothrin, lambda-cyhalothrin, cypermethrin,cyromazine, deltamethrin, diafenthiuron, diazinon, diflubenzuron,dimethoate, diofenolan, emamectin, endosulfan, esfenvalerate, ethiprole,fenothicarb, fenoxycarb, fenpropathrin, fenvalerate, fipronil,flonicamid, flucythrinate, tau-fluvalinate, flufenerim (UR-50701),flufenoxuron, fonophos, halofenozide, hexaflumuron, imidacloprid,indoxacarb, isofenphos, lufenuron, malathion, metaldehyde,methamidophos, methidathion, methomyl, methoprene, methoxychlor,monocrotophos, methoxyfenozide, nithiazin, novaluron, noviflumuron(XDE-007), oxamyl, parathion, parathion-methyl, permethrin, phorate,phosalone, phosmet, phosphamidon, pirimicarb, profenofos, pymetrozine,pyridalyl, pyriproxyfen, rotenone, spinosad, spiromesifin (BSN 2060),sulprofos, tebufenozide, teflubenzuron, tefluthrin, terbufos,tetrachlorvinphos, thiacloprid, thiamethoxam, thiodicarb,thiosultap-sodium, tralomethrin, trichlorfon and triflumuron; fungicidessuch as acibenzolar, azoxystrobin, benomyl, blasticidin-S, Bordeauxmixture (tribasic copper sulfate), bromuconazole, carpropamid, captafol,captan, carbendazim, chloroneb, chlorothalonil, copper oxychloride,copper salts, cyflufenamid, cymoxanil, cyproconazole, cyprodinil,(S)-3,5-dichloro-N-(3-chloro-1-ethyl-1-methyl-2-oxopropyl)-4-methylbenzamide(RH 7281), diclocymet (S-2900), diclomezine, dicloran, difenoconazole,(S)-3,5-dihydro-5-methyl-2-(methylthio)-5-phenyl-3-(phenyl-amino)-4H-imidazol-4-one(RP 407213), dimethomorph, dimoxystrobin, diniconazole, diniconazole-M,dodine, edifenphos, epoxiconazole, famoxadone, fenamidone, fenarimol,fenbuconazole, fencaramid (SZX0722), fenpiclonil, fenpropidin,fenpropimorph, fentin acetate, fentin hydroxide, fluazinam, fludioxonil,flumetover (RPA 403397), flumorf/flumorlin (SYP-L190), fluoxastrobin(HEC 5725), fluquinconazole, flusilazole, flutolanil, flutriafol,folpet, fosetyl-aluminum, furalaxyl, furametapyr (S-82658),hexaconazole, ipconazole, iprobenfos, iprodione, isoprothiolane,kasugamycin, kresoxim-methyl, mancozeb, maneb, mefenoxam, mepronil,metalaxyl, metconazole, metomino-strobin/fenominostrobin (SSF-126),metrafenone (AC375839), myclobutanil, neo-asozin (ferricmethane-arsonate), nicobifen (BAS 510), orysastrobin, oxadixyl,penconazole, pencycuron, probenazole, prochloraz, propamocarb,propiconazole, proquinazid (DPX-KQ926), prothioconazole (JAU 6476),pyrifenox, pyraclostrobin, pyrimethanil, pyroquilon, quinoxyfen,spiroxamine, sulfur, tebuconazole, tetraconazole, thiabendazole,thifluzamide, thiophanate-methyl, thiram, tiadinil, triadimefon,triadimenol, tricyclazole, trifloxystrobin, triticonazole, validamycinand vinclozolin; nematocides such as aldicarb, oxamyl and fenamiphos;bactericides such as streptomycin; acaricides such as amitraz,chinomethionat, chlorobenzilate, cyhexatin, dicofol, dienochlor,etoxazole, fenazaquin, fenbutatin oxide, fenpropathrin, fenpyroximate,hexythiazox, propargite, pyridaben and tebufenpyrad; and biologicalagents including entomopathogenic bacteria, such as Bacillusthuringiensis subsp. Aizawai, Bacillus thuringiensis subsp. Kurstaki,and the encapsulated delta-endotoxins of Bacillus thuringiensis (e.g.,Cellcap, MPV, MPVII); entomopathogenic fungi, such as green muscardinefungus; and entomopathogenic virus including baculovirus,nucleopolyhedro virus (NPV) such as HzNPV, AfNPV and granulosis virus(GV) such as CpGV. Methods of the invention may also comprise the use ofplants genetically transformed to express proteins toxic to invertebratepests (such as Bacillus thuringiensis delta-endotoxins). In suchembodiments, the effect of exogenously applied invertebrate pest controlcompounds may be synergistic with the expressed toxin proteins.

General references for these agricultural protectants include ThePesticide Manual, 13th Edition, Tomlin, Ed., British Crop ProtectionCouncil, Farnham, Surrey, U.K., 2003 and The BioPesticide Manual, 2^(nd)Edition, Copping, Ed., British Crop Protection Council, Farnham, Surrey,U.K., 2001.

In certain instances, combinations with other invertebrate pest controlcompounds or agents having a similar spectrum of control but a differentmode of action will be particularly advantageous for resistancemanagement. Thus, compositions of the present invention can furthercomprise a biologically effective amount of at least one additionalinvertebrate pest control compound or agent having a similar spectrum ofcontrol but a different mode of action. Contacting a plant geneticallymodified to express a plant protection compound (e.g., protein) or thelocus of the plant with a biologically effective amount of a compound ofthis invention can also provide a broader spectrum of plant protectionand be advantageous for resistance management.

Thus, methods of the invention employ a herbicide or herbicidecombination and may further comprise the use of insecticides and/orfungicides, and/or other agricultural chemicals such as fertilizers. Theuse of such combined treatments of the invention can broaden thespectrum of activity against additional weed species and suppress theproliferation of any resistant biotypes.

Methods of the invention can further comprise the use of plant growthregulators such as aviglycine, N-(phenylmethyl)-1H-purin-6-amine,ethephon, epocholeone, gibberellic acid, gibberellin A₄ and A₇, harpinprotein, mepiquat chloride, prohexadione calcium, prohydrojasmon, sodiumnitrophenolate and trinexapac-methyl and plant growth modifyingorganisms such as Bacillus cereus strain BP01.

Embodiments of the present invention are further defined in thefollowing Examples. It should be understood that these Examples aregiven by way of illustration only. From the above discussion and theseExamples, one skilled in the art can ascertain the essentialcharacteristics of this invention, and without departing from the spiritand scope thereof, can make various changes and modifications of theembodiments of the invention to adapt it to various usages andconditions. Thus, various modifications of the embodiments of theinvention, in addition to those shown and described herein, will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

EXPERIMENTAL

The following abbreviations are used in describing the presentinvention.

-   -   ALS acetolactate synthase protein    -   bp base pair    -   glyat4621 glyphosate acetyltransferase gene    -   GLYAT4621 glyphosate acetyltransferase protein    -   zm-als wild type acetolactate synthase gene from brassica    -   zm-hra modified version of acetolactate synthase gene from        brassica    -   kb kilobase    -   PCR polymerase chain reaction    -   UTR untranslated region

Example 1 Insert and Flanking Border Sequence Characterization ofBrassica Event DP0-73496-4

Brassica (Brassica napus L.) has been modified by the insertion of theglyphosate acetyltransferase gene (glyat4621) derived from Bacilluslicheniformis and optimized by gene shuffling. Plasmid PHP28181 containsan expression cassette as further described hereafter.

DNA construct PHP28181 was made by cloning the GAT4621:PINII TERMfragment excised from DNA construct pZSL149 with BamHI and Mfel doubledigestion downstream to the AT-UBQ10 promoter of DNA construct QC272 inthe same BamHI and Mfel sites using T4 DNA ligase (New England Lab). Theresulting PHP28181 DNA contains the expression cassette: AT-UBQ10(DUPONT) PRO:GAT4621:PINII TERM. See, FIG. 1 and FIG. 2.

The 2112 bp PHP28181A DNA fragment was prepared from plasmid PHP28181with HindIII and NotI restriction enzyme double digestion. The digestedplasmid DNA was resolved in a 1% agarose gel by electrophoresis. The DNAband of the correct size was excised and DNA fragment was extractedusing a Qiagen DNA fragment extraction kit (Qiagen). DNA fragment puritywas checked by PCR with a series of dilutions of amp+ positive controlDNA since the PHP28181 plasmid contains an amp+ gene in its backbone.DNA fragment concentration was measured spectrophotometrically andconfirmed by comparing to DNA low mass markers (InVitrogen) in anagarose gel.

Transformation was accomplished essentially as described in Chen andTulsieram, US Patent Application Publication Number 2007/0107077. Budswere collected from donor line NS1822BC and sterilized. Buds were thenhomogenized, filtered, and washed to collect the microspores. Theresultant microspore suspension was adjusted to a specified density andcultured for 2 days. Embryogenic microspores were then isolated viagradient centrifugation and cultured.

Gold particles coated with the PHP28181A DNA fragment were used fortransformation. Biolistic transformation was carried out using thePDS-1000/He Particle Delivery System (Bio-Rad, Hercules, Calif.) asdescribed by Klein, et al., (1987) Nature 327:70-73. Transformedembryogenic microspores were cultured in fresh medium in dark conditionsfor 10-12 days, then under dim light for 1-3 weeks. Green embryos weretransferred to fresh medium and cultured for two weeks to select forglyphosate tolerance. Germinated shoots and/or plants were transferredto growth medium supplemented with glyphosate.

The glyat4621 gene was derived from the soil bacterium Bacilluslicheniformis and was synthesized by a gene shuffling process tooptimize the acetyltransferase activity of the GLYAT4621 enzyme (Castle,et al., (2004) Science 304:1151-1154). The inserted fragment (FIG. 3)from this plasmid contains the glyat4621 gene cassette. The expressionof the glyat4621 gene is controlled by the UBQ10 regulatory region fromArabidopsis and the pinII terminator (see, Table 4). A summary of thetransformation fragment of plasmid PHP28181 is shown in Table 4. Thegenetic elements of plasmid PHP28181 used in the creation of DP-073496-4are shown in Table 3.

TABLE 3 Description of Genetic Elements in Plasmid PHP28181 Location onKnown Size plasmid (base Genetic (base Region pair position) Elementpairs) Description Transformation   1 to 2112 2112 See Table 4 forinformation on Fragment the elements in this region PHP28181A Plasmid2113 to 4770 includes 2658 DNA from various sources for Constructelements plasmid construction and below plasmid replication 2736 to 3596bla (Ap^(R)) 861 β-lactamase gene coding for ampicillin resistance fromE. coli (Sutcliiffe, 1978) (Yanisch-Perron, et al., 1984) 4170 to 4539colE1 ori 370 Hae II fragment containing bacterial origin of replicationregion (colE1 derived) (Tomizawa et al., 1977)

TABLE 4 Description of Genetic Elements in the Transformation FragmentPHP28181A Location on Fragment Size (base pair Genetic (base position)Element pairs) Description 1 to 7 Polylinker 7 Region required forcloning genetic elements Region   8 to 1312 UBQ10 1305 Version of thepromoter region from Arabidopsis Promoter thaliana UBQ10 polyubiquitingene (Norris et al., 1993) developed by the E. I. duPont de Nemours andCompany 1313 to 1335 Polylinker 23 Region required for cloning geneticelements Region 1336 to 1779 gat4621 Gene 444 Synthetic glyphosateN-acetyltransferase gene (Castle et al., 2004; Siehl et al., 2007) 1780to 1796 Polylinker 17 Region required for cloning genetic elementsRegion 1797 to 2106 pinII 310 Terminator region from Solanum tuberosumproteinase Terminator inhibitor II gene (Keil et al., 1986; An et al.,1989) 2107 to 2112 Polylinker 6 Region required for cloning geneticelements Region

The nucleotide sequence of the inserted DNA in the DP-073496-4 event hasbeen determined. PCR amplification of the unique junctions spanning theintroduced genetic elements can distinguish DP-073496-4 plants fromtheir non-genetically-modified counterparts and can be used to screenfor the presence of the inserted DNA, even at very low concentrations.Described below is a construct-specific polymerase chain reaction (PCR)assay on genomic DNA from DP-073496-4 Brassica.

Specifically, genomic DNA from the test substance (plant material ofevent DP-073496-4) and the control substance (plant material of anon-genetically modified Brassica with a genetic backgroundrepresentative of the event background) is isolated and subjected toqualitative PCR amplification using a construct-specific primer pair.The PCR products are separated on 1.5% or 2% agarose gels to confirm thepresence of the inserted construct in the genomic DNA generated from thetest substance, and absence in the genomic DNA generated from thecontrol substance. A reference standard (100 base pair DNA Ladder;Invitrogen Corporation Catalog #10380-012) is used to determine the PCRproduct size.

Test and control samples are harvested from plants. Genomic DNAextraction from the test and control tissues is performed using astandard urea extraction protocol, if leaf tissue. Genomic DNA from thetest and control samples is isolated using Wizard® Magnetic 96 DNA PlantSystem (Promega Corporation Catalog #FF3760), if seed tissue. GenomicDNA is quantified on a spectrofluorometer using PicoGreen® reagent(Molecular Probes, Inc., Eugene, Oreg.) and/or visualized on an agarosegel to confirm quantitation values and to determine the DNA quality.

Genomic DNA isolated from plant material of event DP-073496-4 andcontrol samples is subjected to PCR amplification (PCR Master MixCatalog #7505 from Promega Corporation) utilizing a construct-specificprimer pair which spans at least a portion of the glyat4621 codingregion, and allows for the unique identification of maize eventDP-073496-4. A second primer set is used to amplify an endogenous geneas a positive control for PCR amplification. The PCR target site andsize of the expected PCR product for each primer set are compared to theobserved results.

Example 2 Characterization of Event DP-073496-4 by Southern Blot

Southern blot analyses (Southern, 1975) are performed to investigate thenumber of sites of insertion of the transforming DNA, the copy numberand functional integrity of the genetic elements and the absence ofplasmid backbone sequences.

The method used is described generally as follows. Genomic DNA isextracted from lyophilized tissue sampled from DP-073496-4 Brassica andnon-genetically-modified control plants. Genomic DNA is digested withrestriction endonuclease enzymes and size-separated on an agarose gel. Amolecular weight marker is run alongside samples for size estimationpurposes. DNA fragments separated on agarose gel are depurinated,denatured and neutralized in situ and transferred to a nylon membrane.Following transfer to the membrane, the DNA is bound to the membrane byUV crosslinking. Fragments homologous to the glyat4621 gene aregenerated by PCR from plasmid PHP28181, separated on an agarose gel bysize, exsized and purified using a gel extraction kit. Labeled probe ishybridized to the target DNA on the nylon membranes for detection of thespecific fragments. Washes after hybridization are carried out at highstringency. Blots are exposed to X-ray film for one or more time pointsto detect hybridizing fragments and visualize molecular weight markers.

Example 3 Expression of the Insert

Expression of the GLYAT4621 protein is evaluated using leaf tissuecollected from transgenic plants. For example, four fresh leaf punchesmay be collected and ground in sample extraction buffer using aGenoGrinder (Spex Certiprep). Total Extractable Protein (TEP) can bedetermined using the Bio-Rad Protein assay, which is based on theBradford dye-binding procedure. Sample extracts may be diluted in sampleextraction buffer for ELISA analysis.

The levels of expression of the GAT4621 protein in DP-073496-4 Brassicacan be determined by quantitative enzyme linked immunosorbent assay(ELISA) of samples obtained from multiple field trial locations.Replicate seed samples (three replicates) may be obtained fromDP-073496-4 plants treated with the maximum recommended label rate ofTouchdown® Total glyphosate herbicide (500 g/l glyphosate as potassiumsalt; 0.60-1.35 l/ha), applied at the cotyledon to 6-leaf stage, as thisrepresents a likely commercial cultivation scenario.

Another way to verify the expression of the insert in DP-073496-4Brassica plants is to evaluate the transformed plants' tolerance toglyphosate. Multigenerational stability and within-generationsegregation of the herbicide tolerant trait conferred by expression ofthe GAT4621 enzyme will be confirmed using a functional assay forherbicide tolerance. Tests are conducted on at least three generationsof plant material. Herbicide injury may be scored as described in Table5.

TABLE 5 The 0 to 100 crop response rating system for herbicide injuryMain Rating categories Detailed description 0 No Effect No cropreduction or injury 10 Slight Effect Slight crop discoloration orstunting 20 Some crop discoloration, stunting, or stunt loss 30 Cropinjury more pronounced, but not lasting 40 Moderate Moderate injury,crop usually recovers 50 Effect Crop injury more lasting, recoverydoubtful 60 Lasting crop injury, no recovery 70 Severe Effect Heavy cropinjury and stand loss 80 Crop nearly destroyed - A few surviving plants90 Only occasional live crop plants left 100 Complete Complete cropdestruction Effect

Example 4 Construct Specific PCR Analysis of Brassica Event DP-073496-4

Genomic DNA isolated from leaf of DP-073496-4 canola (T2F2 generation)and control canola (non-genetically modified) was subjected to PCRamplification (Roche High Fidelity PCR Master Kit, Roche Catalog#12140314001) utilizing the construct-specific primer pair(09-0-3290/09-0-3288) which spans the ubiquitin promoter and the gat4621gene cassette (FIG. 4). A second primer set (09-0-2812/09-0-2813) wasused to amplify the endogenous canola FatA gene as a positive controlfor PCR amplification. The PCR target site and size of the expected PCRproduct for each primer sets are shown in Table 8. PCR reagents andreaction conditions are shown in Table 9. The primer sequences used inthis study are listed in Table 10. In this study, 100 ng of leaf genomicDNA was used in all PCR reactions.

A PCR product of approximately 600 bp in size amplified by theconstruct-specific primer set 09-0-3290/09-0-3288 was observed in PCRreactions using plasmid PHP28181 (10 ng) as a template and threeDP-073496-4 canola plants, but absent in three control canola plants andthe no-template control (FIG. 5). Samples were loaded as shown in Table6.

TABLE 6 Lane Sample 1 Low Mass Molecular Weight Marker 2 Blank 3Non-Genetically Modified canola C1 4 Non-Genetically Modified canola C25 Non-Genetically Modified canola C3 6 DP-073496-4 canola T1 7DP-073496-4 canola T2 8 DP-073496-4 canola T3 9 NT Control 10 PlasmidPHP28181 11 Blank 12 Low Mass Molecular Weight Marker

These results correspond with the expected PCR product size (675 bp) forsamples containing DP-073496-4 canola genomic DNA. A PCR productapproximately 450 bp in size was observed for both DP-073496-4 canolaand control canola plants following PCR reaction with the primer set09-0-2812/09-0-2813 for detection of the endogenous FatA gene (FIG. 6).Samples were loaded as shown in Table 7.

TABLE 7 Lane Sample 1 Low Mass Molecular Weight Marker 2 Blank 3Non-Genetically Modified canola C1 4 Non-Genetically Modified canola C25 Non-Genetically Modified canola C3 6 DP-073496-4 canola T1 7DP-073496-4 canola T2 8 DP-073496-4 canola T3 9 NT Control 10 PlasmidPHP28181 11 Blank 12 Low Mass Molecular Weight Marker

These results correspond with the expected PCR product size (506 bp) forgenomic DNA samples containing the canola endogenous FatA gene. Theendogenous target band was not observed in the no-template control.

TABLE 8 PCR Genomic DNA Target Site and Expected Size of PCR ProductsExpected Size of Primer Set Target Site PCR Product (bp)09-0-3290/09-0-3288 Construct-Specific ubiquitin 675 promoter andgat4621 09-0-2812/09-0-2813 Endogenous canola FatA 506 gene¹ PCR:POLYMERASE CHAIN REACTION BP: BASE PAIRS ¹Genbank accession number forFatA gene is X87842.1. This sequence was used to design PCR primers.

TABLE 9 PCR Reagents and Reaction Conditions PCR Reagents PCR ReactionConditions Volume Cycle Temp Time # Reagent (μL) Element (° C.) (sec)Cycles Template DNA 1 Initial 94 120 1 (100 ng/μl) Denaturation Primer 1(10 μM) 0.75 Denaturation 94 10 35 Primer 2 (10 μM) 0.75 Annealing 65 20PCR Master Mix* 12.5 Elongation 72 45 ddH₂O 10 Final Elongation 72 180 1Hold Cycle 4 Until analysis PCR: POLYMERASE CHAIN REACTION DDH₂O:DOUBLE-DISTILLED WATER *Roche High Fidelity Master Mix

TABLE 10 List of Primer Sequences Used in PCR Reactions Primer TargetName Sequence 5′-3′ Sequence 09-0-3290 SEQ ID NO 4: UbiquitinAGCTATTGCTTCACCGCCTTAGC Promoter 09-0-3288 SEQ ID NO: 5 gat4621GCTCAGCTTGGTGGAATGAAGCCAC 09-0-2812 SEQ ID NO: 6 CanolaGACACAAGGCGGCTTCAAAGAGTTACAGATG Endo- genous FatA 09-0-2813 SEQ ID NO 7:Canola ACAATGTCATCTTGCTGGCATTCTCTTCTG Endo- genous FatA

Example 5 Further Insert and Flanking Border Sequence Characterizationof Brassica Event DP-073496-4

To characterize the integrity of the inserted DNA and the genomicinsertion site, the flanking genomic DNA border regions of theDP-073496-4 event were determined. Flanking genomic sequence ofDP-073496-4 is included within SEQ ID NO: 2. PCR amplification from theinsert and border sequences confirms that the border regions are ofBrassica origin and that the junction regions can be used foridentification of DP-073496-4 Brassica. Overall, characterization of theinsert and genomic border sequences, along with Southern blot data,indicate a single insertion of the DNA fragment present in the Brassicagenome. Various molecular techniques are then used to specificallycharacterize the integration site.

In the initial characterization, the flanking genomic border regions arecloned and sequenced using the GenomeWalker and inverse PCR methods.Using information from the flanking border sequence, PCR is performed onDP-073496-4 genomic DNA and unmodified control genomic DNA. Thoseskilled in the art will also include a control PCR using an endogenousgene to verify that the isolated genomic DNA is suitable for PCRamplification.

TABLE 11 PCR-based event-specific detection methods PCR Assay Primer 1Primer 2 Probe event type Name Sequence Name Sequence Name Sequence 5′label Quencher DP- Gel- 10-O- GGTCCGTGGGC 10-O- TTATCCGGTCCTAG — — — —φ73496- based 3514 CTTCCTAAACGT 3515 ATCATCAGTTCATA 4 SEQ ID GCCG SEQ IDCAAACCTCC NO: 20 NO: 23 DP- Real- 09-O- GTTCTTCTCTTC 09-O- CAAACCTCCATAG09-QP83 TTAGTTAGATC FAM MGB φ73496- time 2824 ATAGCTCATTAC 2825AGTTCAACATCTTA SEQ ID AGGATATTCTT 4 SEQ ID AGTTTT SEQ ID A NO: 26 GNO: 21 NO: 24 FatA A- Real- 09-O- ACAGATGAAGT 09-O- CAGGTTGAGATCC09-QP87 AAGAAGAATCA FAM MGB specific time 3249 TCGGGACGAGT 3251ACATGCTTAAATAT SEQ ID TCATGCTTC SEQ ID AC SEQ ID NO: 27 NO: 22 NO: 25  

TABLE 12 Summary Table of SEQ ID NOS SEQ ID NO Description  1GAT4621 protein  2 DP-073496-4 insert and flanking sequence  3 PHP28181A 4 Primer 09-0-3290 (SEQ ID NO: 4 AGCTATTGCTTCACCGCCTTAGC)Target - Ubiquitin Promoter  5 Primer 09-0-3288 (SEQ ID NO: 5GCTCAGCTTGGTGGAATGAAGCCAC) Target gat4621  6Primer 09-0-2812 (SEQ ID NO: 6 GACACAAGGCGGCTTCAAAGAGTTACAGATG) TargetCanola Endogenous FatA  7 Primer 09-0-2813 (SEQ ID NO:ACAATGTCATCTTGCTGGCATTCTCTTCTG) Canola Endogenous FatA  8Right border genomic sequence  9 Left border genomic sequence 10Complete internal transgene 11 Complete flanking and internal transgene12 Right flanking genomic/right border transgene (10 nt/10 nt) 13Left flanking genomic/left border transgene (10 nt/10 nt) 14Right flanking genomic/right border transgene (20 nt/20 nt) 15Left flanking genomic/left border transgene (20 nt/20 nt) 16Right flanking genomic/right border transgene (30 nt/30 nt) 17Left flanking genomic/left border transgene (30 nt/30 nt) 18Right flanking genomic/complete transgene 19Left flanking genomic/complete transgene 20 Primer 10-O-3514 21Primer 09-O-2824 22 Primer 09-O-3249 23 Primer 10-O-3515 24Primer 09-O-2825 25 Primer 09-O-3251 26 Primer 09-QP83 27 Primer 09-QP87

The article “a” and “an” are used herein to refer to one or more thanone (i.e., to at least one) of the grammatical object of the article. Byway of example, “an element” means one or more element.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

The invention claimed is:
 1. A method for identifying event DP-073496-4in a biological sample, comprising (a) contacting said sample with afirst and a second primer, wherein at least one of said first and saidsecond primer targets a DP-073496-4 specific region; and, (b) amplifyinga polynucleotide comprising the DP-073496-4 specific region wherein theDP-073496-4 specific region comprises a junction sequence selected fromthe group consisting of SEQ ID NO: 12 and
 13. 2. The method of claim 1,wherein the polynucleotide comprising the DP-073496-4 specific regioncomprises SEQ ID NO: 14, or
 16. 3. The method of claim 1, wherein thepolynucleotide comprising the DP-073496-4 specific region comprises SEQID NO: 15, or
 17. 4. The method of claim 1, further comprising detectingthe DP-073496-4 specific region.
 5. The method of claim 1, wherein theamplification of a polynucleotide comprising the DP-073496-4 specificregion indicates seed purity or seed lot purity.
 6. The method of claim4, wherein the detection of said DP-073496-4 specific region indicatesseed purity or seed lot purity.
 7. The method of claim 1, wherein thefirst and the second primer can amplify a sequence comprising SEQ ID NO:12, 13, 14, 15, 16 or 17.